Nor-1 and nur77 nuclear receptors as targets for anti-leukemia therapy

ABSTRACT

The present invention is directed to the application of nuclear receptor transcription factors as molecular targets for therapeutic intervention in the treatment of myeloid leukemia. More specifically, nor-1 and nur77 nuclear receptors are targets for myeloid leukemia therapy.

[0001] This patent application claims priority to U.S. ProvisionalApplication, Serial No. 60/373,238, filed Apr. 17, 2002, which isincorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The present invention was developed using funds from NIH GrantNo. DK57743. The United States Government may have certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The field of the present invention generally includes cellbiology, molecular biology, and cancer therapy, such as for leukemia.More particularly, the present invention regards nor-1 and nur77 nuclearreceptors as targets for anti-leukemic drug intervention.

BACKGROUND OF THE INVENTION

[0004] Leukemia is a type of cancer that is defined as an excessiveproduction of cells of bone marrow origin (hematopoietic cells).Leukemia is further classified based on the specific cell lineage thatis affected (lymphoid and myeloid). The detrimental effects of theuncontrolled production of these cells include altered development ofother hematopoietic cell lineages and infiltration into peripheraltissues, such as the lung, that can affect the normal function of thatorgan, and may ultimately lead to death.

[0005] Nor-1 (NR4A3, TEC, MINOR, CHN) and nur77 (NR4A1, TR3, NGFI-B,NAK1, HMR) are members of the Nuclear Receptor Superfamily. Nuclearreceptors are transcription factors that are activated by binding smallmolecule ligands. Ligand binding induces conformational changes innuclear receptors that allow them to recruit coregulator proteins to thetranscription apparatus to induce transcription of specific genes.Transcriptional regulation activity of nor-1 and nur77 can also beregulated by binding of co-factors, or posttranslational modificationssuch as phosphorylation induced by signaling cascades as a result ofcell exposure to stimuli such as but not limited to growth factors,neurotransmitters, cyclic AMP, cytokines, or mechanical stimulation. Onewell-studied area where nor-1 and nur77 have been thought to play a roleis in the development of t-lymphocytes. T-lymphocytes originate in thebone marrow and then migrate to the thymus where they undergo themajority of their maturation. It is known that nor-1 and nur77 arenecessary for a process called negative selection within the thymus.Negative selection causes t-lymphocytes reactive to self-proteins to diebefore they enter the periphery, and it occurs late in t-lymphocytedevelopment. This process is important in preventing autoimmunity.

[0006] Wu et al. (2002) describes interaction and inhibition of Nur77 bythe promyelocytic leukemia protein (PML) in a dose-dependent manner.Specifically, the coiled-coil domain of PML interacts with theDNA-binding domain of Nur77 (amino acids 267-332). The data is describedin the context of supporting a role for PML/Nur77 interaction inregulating cell growth and apoptosis.

[0007] Bandoh et al. (1997) demonstrate that mechanical agitationtransiently induced nor-1, ngfi-b (nur77), and nurr1 mRNAs in severalleukemic cell lines in a dose-dependent manner, particularly in theHL-60 promyelocytic leukemia cell line.

[0008] Thus, during hematopoiesis, prior to the negative selection stageof t-lymphocytes, no role of lymphocyte development has been assigned toeither nor-1 or nur77. The present invention addresses such a findingand provides methods and compositions useful for leukemia prevention andtherapy.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention is directed to methods and compositionsrelated to nuclear receptors nor-1 and/or nur77 for therapy andprevention of leukemia, particularly myeloid leukemia withdifferentiation.

[0010] In an embodiment of the present invention, there is a method ofinhibiting proliferation of a hematopoietic cell, comprising the step ofmodulating the level of nor-1 and/or nur77 nuclear receptor. In someembodiments, the hematopoietic cell may be a hematopoietic stem cell ora hematopoietic myeloid cell. The modulating step may be defined asincreasing the level of a nor-1 and/or nur77 nuclear receptorpolypeptide, and the increasing step may be defined as increasing thelevel of a nor-1 and/or nur77 nuclear receptor polynucleotide.

[0011] In some embodiments, the nor-1 and/or nur77 nuclear receptorpolynucleotide is increased through administration of a vectorcomprising the polynucleotide, and the vector may be a viral vector or anon-viral vector. Viral vector includes an adenoviral vector, aretroviral vector, or an adeno-associated vector. In a specificembodiment, the viral vector is an adenoviral vector. In anotherspecific embodiment, the non-viral vector is a plasmid. In anotherspecific embodiment, the nor-1 and/or nur77 nuclear receptorpolynucleotide is increased through upregulation of expression. In afurther specific embodiment, the upregulation of expression is of thenor-1 and/or nur77 nuclear receptor, and the upregulation of expressionof the nor-1 and/or nur77 nuclear receptor may be through administrationof growth factors, cytokines, cyclic AMP, or a mixture thereof. In someembodiments, the cell is in a mammal afflicted with leukemia.

[0012] In another embodiment of the present invention, there is a methodof inhibiting proliferation of a hematopoietic cell, comprising the stepof modulating the activity of a nor-1 and/or nur77 nuclear receptor. Thehematopoietic cell may be a hematopoietic stem cell or a hematopoieticmyeloid cell. In some embodiments, the modulating step is defined asincreasing transcriptional activity of a nor-1 and/or nur77 nuclearreceptor polypeptide. In other embodiment of the present invention, themodulating step is further defined as administering an agonist to thenor-1 and/or nur77 nuclear receptor polypeptide.

[0013] In an additional embodiment of the present invention, there is amethod of treating leukemia in an individual, comprising the step ofmodulating a nor-1 and/or nur77 nuclear receptor in the individual. In aspecific embodiment, the modulating step occurs in a hematopoietic cellof the individual. The hematopoietic cell may be a hematopoietic stemcell or a hematopoietic myeloid cell.

[0014] In an additional embodiment of the present invention, themodulating step is further defined as increasing the activity of a nor-1and/or nur77 nuclear receptor polypeptide, is further defined asincreasing the level of a nor-1 and/or nur77 nuclear receptorpolypeptide, or is further defined as increasing the level of a nor-1and/or nur77 nuclear receptor polynucleotide.

[0015] In a further specific embodiment of the present invention, theincreasing activity step is further defined as introducing an agonist tosaid nor-1 and/or nur77 nuclear receptor polypeptide. In someembodiments, the introducing step is further defined as administeringsaid agonist in a pharmaceutically acceptable composition to saidindividual, such as a ligand of said nor-1 and/or nur77 nuclearreceptor, although the agonist may not be a ligand of said nor-1 and/ornur77 nuclear receptor.

[0016] In some embodiments, the increasing the level of a nor-1 and/ornur77 nuclear receptor polynucleotide step is defined as increasingexpression of a respective nor-1 and/or nur77 nuclear receptor in a cellof the individual. The cell may be a hematopoietic bone marrow stem cellor a hematopoietic myeloid cell. In an additional specific embodiment,the increasing the level of a nor-1 and/or nur77 nuclear receptorpolynucleotide step is defined as increasing the half-life of arespective nor-1 and/or nur77 nuclear receptor mRNA in a cell of theindividual. In a further specific embodiment, the method furthercomprises the step of administering said cell to an individual.

[0017] In an additional embodiment of the present invention, there is amethod of increasing the level of a nor-1 and/or nur77 nuclear receptorin a hematopoietic cell, comprising the step of administering a compoundto the cell to increase the expression of said nor-1 and/or nur77nuclear receptor. The compound may be a growth factor, cytokine, cyclicAMP, or a mixture thereof. The method may be further defined asadministering said compound in a pharmaceutically acceptable compositionto said individual.

[0018] In another embodiment of the present invention, there is a methodof identifying an upregulator of expression of a nor-1 and/or nur77nuclear receptor, comprising the steps of introducing to a cell a testagent, wherein the cell comprises a marker sequence and wherein theexpression of the marker sequence is regulated by a nor-1 and/or nur77nuclear receptor regulatory sequence; and measuring for an increase inthe expression level of the marker sequence, wherein when the increaseoccurs following introduction of said test agent to the cell, the testagent is the upregulator. In a specific embodiment of the presentinvention, the method further comprises administering the upregulator ina pharmaceutically acceptable composition to an individual. In anotherspecific embodiment, the individual is susceptible to leukemia or isdiagnosed with leukemia.

[0019] In an additional embodiment of the present invention, there is amethod of identifying a compound for the treatment of leukemia,comprising the steps of obtaining a compound suspected of havingactivity of a nor-1 and/or nur77 nuclear receptor agonist; anddetermining whether said compound has said activity. In a specificembodiment, the agonist is a ligand of a nor-1 and/or nur77 nuclearreceptor. The method may further comprise dispersing the compound in apharmaceutical carrier; and administering a therapeutically effectiveamount of the compound in the carrier to an individual having leukemia.

[0020] In an additional embodiment of the present invention, there is acompound obtained by a method described herein.

[0021] In another embodiment of the present invention, there is apharmacologically acceptable composition comprising the compoundobtained by a method described herein and a pharmaceutical carrier.

[0022] In an additional embodiment of the present invention, there is amethod of screening for a compound for the treatment of leukemia,comprising the steps of providing a first vector comprising a nor-1 ornur77 nucleic acid sequence encoding a respective nor-1 or nur77 geneproduct, wherein the expression of said nor-1 or nur77 nucleic acidsequence is under the control of a first regulatory sequence; providinga second vector comprising a reporter nucleic acid sequence encoding areporter gene product, wherein the expression of said reporter nucleicacid sequence is under the control of a second regulatory sequence,wherein the second regulatory sequence is responsive to nor-1 or nur77;providing a test agent; providing a leukemia cell line, wherein cells insaid cell line comprise conditions suitable for expression of said nor-1or nur77 gene product and said reporter gene product; and assayingtranscriptional regulation activity of said nor-1 or nur77 gene productby measuring expression or activity of the reporter gene product in thepresence of said test agent, wherein when the expression or activity ofthe reporter gene product changes in the presence of the test agent, thetest agent is the compound for the treatment of leukemia. The leukemiccell line may be K562, U937, AML-193, HL-60, LSTRA, or CEM. In aspecific embodiment, the first vector, second vector, test agent, or acombination thereof are introduced into the cell line. In anotherspecific embodiment, the reporter nucleic acid is β-galactosidase, greenfluorescent protein, blue fluorescent protein, or chloramphenicolacetyltransferase. The expression or activity of the reporter geneproduct increases in the presence of the test agent, in someembodiments.

[0023] In an additional embodiment of the present invention, there is amouse model for leukemia, comprising a mouse having defective nor-1and/or nur77 nucleic acid sequences. In a specific embodiment, the mouseis further defined as having a knockout mutation in the genes encodingnor-1 and nur77, respectively. In another specific embodiment, the mouseis further defined as having the nor-1KO/nur77± genotype, thenor-1±/nur77 KO, or the nor-1KO/nur77KO, wherein KO is defined as aknockout. In a further specific embodiment, the mouse comprises at leastone symptom of leukemia.

[0024] The foregoing has outlined rather broadly the features andtechnical advantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] For a more complete understanding of the present invention,reference is now made to the following descriptions taken in conjunctionwith the accompanying drawing, in which:

[0026]FIG. 1 depicts a growth curve representing several litters thatwere weighed daily for a period of 14 days.

[0027]FIG. 2 illustrates lymphadenopathy and splenomegaly observed inthe nor-1KO (knockout)/nur77KO mice. Tissues on the right in both panelsare from normal littermates.

[0028]FIG. 3 is an illustration of liver discoloration observed in thenor-1KO/nur77KO mice (right side is normal littermate).

[0029]FIG. 4 illustrates altered histology of the spleen and thymus inthe nor-1KO/nur77KO mice. Left panels show tissue from normallittermates.

[0030]FIG. 5 shows abnormal presence of medullary epithelial cellsthroughout the nor-1KO/nur77KO thymus (right panel shows normal thymus).

[0031]FIG. 6 demonstrates the total number of thymocytes are reduced inthe nor-1KO/nur77KO mice.

[0032]FIG. 7 illustrates reduction in total thymocyte number is notlimited to any specific CD4/CD8 developmental stage.

[0033]FIG. 8 shows perivascular cellular infiltrates in the liver, lung,and pancreas of the nor-1KO/nur77KO mice. The left hand panels showtissues from normal littermates.

[0034]FIG. 9 demonstrates CD11b/Gr-1 expressing cells are increased inthe nor-1KO/nur77KO lymphoid tissues and blood. Dotplots from normallittermates are shown on the left.

[0035]FIG. 10 shows positive myeloperoxidase staining in thenor-1KO/nur77KO perivascular cellular infiltrates and lymphoid tissues.

[0036]FIG. 11 shows cells within the perivascular infiltration andlymphoid tissue in the nor-1KO/nur77KO mice are CD11b positive.

[0037]FIG. 12 demonstrates abnormal hematopoiesis in the bone marrow ofthe nor-1KO/nur77KO mice (the left panels are bone marrow results fromnormal littermates.)

[0038]FIG. 13 shows hypoallelic nor-1KO/nur77± mice display abnormallymphoid tissue architecture.

[0039]FIG. 14 demonstrates perivascular cell infiltrates in thehypoallelic nor-1KO/nur77± mouse.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions

[0041] As used herein the specification, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

[0042] The term “agonist” as used herein is defined as a factor thatpromotes, facilitates or enhances the activity or function of anotherbiological entity. In a specific embodiment, the agonist is an agonistof transcription regulatory activity of the nor-1 or nur77 polypeptide.The agonist may be a small molecule, an amino acid sequence, a nucleicacid sequence, a lipid, a sugar, a carbohydrate, polypeptide, or acombination thereof.

[0043] The term “anti-leukemic activity” as used herein is defined ashaving activity that improves, at least in part, one or more symptoms ofmyeloid leukemia. Symptoms are well known in the art, however, someexamples include excessive production of cells of bone marrow origin(hematopoietic cells) of the myeloid lineage, altered development ofother hematopoietic cell lineages and/or infiltration into peripheraltissues, anemia, and splenomegaly.

[0044] The term “gene product” as used herein is defined as a mRNA, apolypeptide or both an mRNA or polypeptide encoded by a nucleic acidsequence.

[0045] The term “ligand” as used herein is defined as a molecule thatbinds to another molecule, preferably a receptor, and more preferably anuclear-localized receptor. In a specific embodiment, a ligand thatbinds to nor-1 and/or nur77 is preferred. One skilled in the artrecognizes that a ligand includes the whole ligand, or any part or anymutant thereof that remains capable of binding to nor-1 and/or nur77.

[0046] The term “modulating” as used herein is defined as altering thelevel, activity, or both of nor-1 and/or nur77 nuclear receptorpolypeptide.

[0047] The term “non-ligand agonist” as used herein is defined as anagonist that does not directly bind the receptor but enhances itsbiological activity by either increasing the cellular level of nor-1and/or nur77 or activation of nor-1 and/or nur77 protein by covalentmodification such as phosphorylation.

[0048] The term “therapeutically effective” as used herein is defined asthe amount of a compound required to improve some symptom associatedwith a disease. For example, in the treatment of leukemia, a compoundthat decreases, prevents, delays or arrests any symptom of the diseasewould be therapeutically effective. A therapeutically effective amountof a compound is not required to cure a disease. A compound is to beadministered in a therapeutically effective amount if the amountadministered is physiologically significant. A compound isphysiologically significant if its presence results in technical changein the physiology of a recipient organism.

[0049] The term “upregulator” as used herein is defined as a compoundthat indirectly or directly causes an increase in expression of nor-1and/or nur77 nuclear receptors.

[0050] The Present Invention

[0051] Nor-1 and nur77 nuclear receptors are redundant in the process ofnegative selection in the thymus. Therefore, deletion of either of thesegenes in mice does not result in altered t-lymphocyte development. Theinventors predicted based on previous studies that deletion of bothnor-1 and nur77 would result in a defect in the later stages oft-lymphocyte development, specifically negative selection. Unexpectedly,as shown herein, upon deletion of both of these nuclear receptors, micedo not survive past 4 weeks of age. Also unexpectedly, in addition to adefect during the stage of negative selection, lymphocyte development isalso altered during the earlier stages. This early defect in lymphocytedevelopment is secondary to a severe overproduction of myeloid cells inthe bone marrow leading to myeloid leukemia with differentiation in themice lacking both nor-1 and nur77 (nor-1KO/nur77KO). No previous reportshave implicated either nor-1 or nur77 in bone marrow hematopoiesis inthe myeloid lineage or in the prevention of development of leukemia. Theresults that support the diagnosis of myeloid leukemia in these mice aresummarized herein and reflect the novel aspect of the present inventionregarding leukemia prevention and treatment.

[0052] It is an object of the present invention to relate methods oftreatment, methods of prevention, agonists and other compositions tonor-1 and/or nur77 for leukemia. In one aspect of the present invention,both nor-1 and nur77 are within the scope of the present invention,particularly given the striking structurally and genetically relatedredundancy of these two family members. A skilled artisan recognizesthat these genes may also be referred to as being in the Nur nuclearreceptor superfamily or the NGFI-B subfamily of a nuclear receptorsuperfamily. Characteristics of nor-1 and/or nur77 may include a centralDNA binding domain comprising two highly conserved zinc finger motifs(Berg, 1989; Klug and Schwabe, 1995), a ligand-binding domain comprising8-9 heptad repeats of hydrophobic amino acids in the carboxyl terminus,and/or a variable amino-terminal region.

[0053] One skilled in the art recognizes that within the scope of theinvention a NOR-1 sequence is utilized. Examples of nucleic acid NOR-1sequences comprise SEQ ID NO: 1 (1651190), SEQ ID NO: 2 (D38530). SEQ IDNO: 3 (AF050223), SEQ ID NO: 4 (BG235965), SEQ ID NO: 5 (BE65671 1), SEQID NO: 6 (AJ011768), SEQ ID NO: 7 (E14965; a useful exemplary Nor1promoter region), SEQ ID NO: 8 (AJ011767), SEQ ID NO: 9 (D85244, anotherexemplary Nor1 promoter region), SEQ ID NO: 10 (D85243), SEQ ID NO: 11(D85242), and SEQ ID NO: 12 (D85241).

[0054] Examples of amino acid NOR-1 sequences comprise SEQ ID NO: 13(7441771), SEQ ID NO: 14 (Q92570), SEQ ID NO: 15 (JC2493), SEQ ID NO: 16(CAA09764), SEQ ID NO: 17 (CAA09763), SEQ ID NO: 18 (BAA31221), and SEQID NO: 19 (BAA28608).

[0055] One skilled in the art recognizes that within the scope of theinvention a NUR77 sequence is utilized. Examples of nucleic acid NUR77sequences comprise SEQ ID NO: 20 (1339917), SEQ ID NO: 21 (12662548),SEQ ID NO: 22 (BF937382), SEQ ID NO: 23 (BE198460), SEQ ID NO: 24(BE047656), SEQ ID NO: 25 (BE047651), SEQ ID NO: 26 (AW988827), SEQ IDNO: 27 (AA461422), SEQ ID NO: 28 (D49728), and SEQ ID NO: 29 (S77154).

[0056] Examples of amino acid NUR77 sequences comprise SEQ ID NO: 30(127819), SEQ ID NO: 31 (128911), SEQ ID NO: 32 (P22829), SEQ ID NO: 33(AAB33999), SEQ ID NO: 34 (AAA42058), and SEQ ID NO: 35 (A37251). Askilled artisan would know how to retrieve sequences from the NationalCenter for Biotechnology Information's Genbank database or commerciallyavailable databases such as the genetic database by Celera Genomics,Inc. (Rockville, Md.).

[0057] In the present invention, the methods are used for treatingand/or preventing leukemia, particularly myeloid leukemia. Examples ofuse in the treatment would be for the improvement of the disease afterits onset or in helping alleviate at least one symptom. The disease isconsidered to be improved if at least one symptom is alleviated, whereinalleviation may be partial or complete. Symptoms to be alleviatedinclude but are not limited to increased white blood cells in theperipheral blood, altered hematopoietic lineages in the bone marrow,anemia, splenomegaly, hematopoietic infiltration into peripheralnon-hematopoietic tissues, etc. An example of use for the prevention ofthe disease would be the use prior to the onset of leukemia, and thus,prevent or delay its onset.

[0058] One specific embodiment of the present invention is a method ofpreventing or treating leukemia comprising the step of modulating nor-1and/or nur77, such as its function or level. In a specific embodiment,nor-1 and/or nur77 receptor polynucleotide is increased, such as byupregulation of its expression or by increase of the mRNA transcription.In another specific embodiment, nor-1 and/or nur77 nuclear receptorpolypeptide level is increased or the activity of nor-1 and/or nur77nuclear receptor polypeptide is enhanced or facilitated, or bothpolypeptide level is increased and activity is enhanced. One skilled inthe art recognizes that there are a variety of ways to increase nor-1and/or nur77 nuclear receptor levels, such as administering to a cellone or more nor-1 and/or nur77 nuclear receptor polypeptides or toupregulate expression of a nor-1 and/or nur77 nuclear receptorpolynucleotide. Furthermore, a skilled artisan recognizes how to enhancethe activity of nor-1 and/or nur77 nuclear receptor polypeptides, suchas by introducing an agonist to the polypeptide, either directly orindirectly. In other embodiments, a nor-1 or nur77 nuclear receptorpolynucleotide is delivered to a cell to increase level of the nor-1 ornur77 nuclear receptor polynucleotide and/or polypeptide, and inspecific embodiments the cell is comprised in an individual.

[0059] In particular embodiments, the expression of the nor-1 and/ornur77 nuclear receptor is upregulated, wherein the upregulation resultsindirectly or directly with inhibiting proliferation of a hematopoieticcell, such as a hematopoietic stem cell, a hematopoietic myeloid cell,or both. In specific embodiments, the upregulation in expression is aresult of administration of a factors such as but not limited to growthfactors, cytokines, cyclic AMP, or a mixture thereof. Examples of growthfactors include but are not limited to epidermal growth factor,hematopoietic stem cell growth factor (SCGF) (such as is described inU.S. Pat. No. 6,541,217, incorporated by reference herein in itsentirety), granulocyte macrophage-colony stimulating factor (GM-CSF),granulocyte-colony stimulating factor (G-CSF), macrophage-colonystimulating factor (M-CSF), tumor necrosis factors (TNF.alpha. andTNF.beta.), transforming growth factors (TGF.alpha. and TGF.beta.), stemcell factor (SCF), platelet-derived growth factors (PDGF), nerve growthfactor (NGF), fibroblast growth factors (FGF), insulin-like growthfactors (IGF-I and IGF-II), growth hormone, interleukin-1,interleukin-2, keratinocyte growth factor, ciliary neurotrophic growthfactor, Schwann cell-derived growth factor, and vaccinia virus growthfactor. Examples of cytokines include but are not limited to IL-1α,IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF,BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, INF-α, IFN-β, and IFN-γ.

[0060] A skilled artisan recognizes that there are a variety of geneproducts that affect expression of nor1 and/or nur77 expression and, insome embodiments, they are utilized in the present invention. Forexample, the af1R gene activates the transcription of nor-1 (Chang etal., 1993).

[0061] In one embodiment of the present invention, there is a method ofscreening for a compound for the treatment of leukemia by providing afirst vector comprising a nor-1 or nur77 nucleic acid sequence encodinga respective nor-1 or nur77 gene product, wherein the expression of saidnor-1 or nur77 nucleic acid sequence is under the control of a firstregulatory sequence; providing a second vector comprising a reporternucleic acid sequence encoding a reporter gene product, wherein theexpression of said reporter nucleic acid sequence is under the controlof a second regulatory sequence, wherein the second regulatory sequenceis responsive to nor-1 or nur77; providing a test agent; providing aleukemia cell line, wherein cells in said cell line comprise conditionssuitable for expression of said nor-1 or nur77 gene product and saidreporter gene product; and assaying transcriptional regulation activityof said nor-1 or nur77 gene product by measuring expression or activityof the reporter gene product in the presence of said test agent, whereinwhen the expression or activity of the reporter gene product changes inthe presence of the test agent, the test agent is the compound for thetreatment of leukemia.

[0062] A skilled artisan recognizes that the leukemic cell line may beany leukemic cell line, although exemplary leukemic cell lines includeK562, U937, AML-193, HL-60, LSTRA, or CEM. In a specific embodiment, thefirst vector, second vector, test agent, or a combination thereof areintroduced into the cell line. In another specific embodiment, thereporter nucleic acid is β-galactosidase, green fluorescent protein,blue fluorescent protein, or chloramphenicol acetyltransferase, althoughthese are only a few exemplary embodiments and one of skill in the artwould know of additional reporter nucleic acid sequences to utilize. Insome embodiments, the expression or activity of the reporter geneproduct increases in the presence of the test agent, although in otherembodiments it decreases.

[0063] In some embodiments, it is envisioned that a DNA or RNA segmentcomprises a nucleic acid sequence to be expressed operatively linked toits associated control sequences or an appropriate alternative. Forexample, the nucleic acid sequence may be operatively linked to asuitable promoter and a suitable terminator sequence. A “promoter” is acontrol sequence that is a region of a nucleic acid sequence at whichinitiation and rate of transcription are controlled. It may containgenetic elements at which regulatory proteins and molecules may bindsuch as RNA polymerase and other transcription factors. The phrases“operatively positioned,” “operatively linked,” “under control,” and“under transcriptional control” mean that a promoter is in a correctfunctional location and/or orientation in relation to a nucleic acidsequence to control transcriptional initiation and/or expression of thatsequence. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

[0064] The construction of such gene/control sequence DNA constructs iswell-known within the art. In particular embodiments, the promoter isCMV. In certain embodiments for introduction, the DNA segment may belocated on a vector, for example, a plasmid vector or a viral vector.The virus vector may be, for example, selected from the group comprisingretrovirus, adenovirus, herpesvirus, vaccina virus, and adeno-associatedvirus. Such a DNA segment may be used in a variety of methods related tothe invention. The vector may be used to deliver a particular nucleicacid sequence to a cell in a gene transfer embodiment of the invention.Also, such vectors can be used to transform cultured cells, and suchcultured cells could be used, inter alia, for the expression of aparticular sequence in vitro.

[0065] For a method described herein wherein a regulatory sequenceresponsive to nor-1, nur77, or both is utilized, a skilled artisanrecognizes how to obtain the sequence by standard means in the art (see,for example, Philips et al., 1997). In particular embodiments, theregulatory sequence responsive to nor-1, nur77, or both comprises NBRE(AAAGGTCA). In other embodiments, the regulatory sequence comprisesNurRE (Philips et al., 1997), or GTGATATTTACCTCCAAATGCCAG (SEQ ID NO:36). The regulatory sequence responsive to nor-1, nur77, or both may bedirectly or indirectly responsive. That is, nor-1 and/or nur77 mayinteract with another gene product prior to interacting with theregulatory sequence. In alternative embodiments, nor-1 and/or nur77interact with the regulatory sequence or direct the activity of anothergene product to do so.

[0066] In the present invention, there is a method of identifying acompound for the treatment of leukemia by obtaining a compound suspectedof having activity of a nor-1 and/or nur77 nuclear receptor agonist anddetermining whether the compound has the activity. For example, acompound suspected of having activity of a nor-1 and/or nur77 agonistmay be a compound present in a pathway in which nor-1 and/or nur77 arealso members. In a specific embodiment, the agonist is a ligand of anor-1 and/or nur77 nuclear receptor. In another specific embodiment, themethod further comprises dispersing the compound in a pharmaceuticalcarrier; and administering a therapeutically effective amount of thecompound in the carrier to an individual having leukemia.

[0067] In an additional embodiment of the present invention, there is amouse model for leukemia, comprising a mouse having defective nor-1and/or nur77 nucleic acid sequences. The nucleic acid sequence(s) may berendered defective by any standard means in the art, but in a specificembodiment the mouse is further defined as having a knockout mutation inthe genes encoding nor-1 and/or nur77, respectively. The term “knockout”as used herein refers to an alteration in a coding sequence whichrenders the gene or gene product encoded by the coding sequencedefective, such as not being expressed. The means to effect a knockoutin a particular gene or nucleic acid sequence are well known in the art.In another specific embodiment, the mouse is further defined as havingthe nor-1KO/nur77± genotype, the nor-1±/nur77 KO, or thenor-1KO/nur77KO, wherein KO is defined as a knockout. In a furtherspecific embodiment, the mouse comprises at least one symptom ofleukemia, described elsewhere herein.

[0068] Screening Assays—Amino Acid Agonists

[0069] In a specific embodiment of the present invention there is amethod of administering an agonist to nor-1 and/or nur77 nuclearreceptor polypeptide. A skilled artisan recognizes that the agonist inone embodiment is a nor-1 and/or nur77 nuclear receptor ligand andenhances nor-1 and/or nur77 nuclear receptor transcriptional activity bybinding to nor-1 and/or nur77 nuclear receptors. In another embodiment,the agonist is a non-ligand agonist. In some embodiments, the non-ligandagonist results in increased activity of nor-1 and/or nur77 nuclearreceptor. A skilled artisan is aware that standard methods are utilizedto screen for compounds that act as an agonist to nor-1 and/or nur77nuclear receptor. For example, compound banks or oligopeptide librariesare screened in a specific embodiment by methods well known in the artfor activity modulating nor-1 and/or nur77 nuclear receptor, such as itstranscriptional activation activity.

[0070] One embodiment of the present invention is a method to administercompounds that affect nor-1 and/or nur77 nuclear receptor structure.Such compounds may include but are not limited to proteins, peptides,nucleic acids, carbohydrates, or other molecules, which upon bindingalter nor-1 and/or nur77 nuclear receptor structure, thereby enhancing,facilitating, or increasing its activity.

[0071] One embodiment of the present invention is a method to administera compound or compounds that affects nor-1 and/or nur77 nuclear receptorfunction. Such compounds may include but are not limited to proteins,nucleic acids, carbohydrates, or other molecules that upon binding (oradministration if a non-ligand agonist) to improve a function of nor-1and/or nur77 nuclear receptor.

[0072] Screening Assays—Nucleic Acid Agonists

[0073] In an embodiment of the present invention there is a method toincrease nucleic acid levels of nor-1 and/or nur77 nuclear receptor. Anexample presented herein provides a substance that is a candidate forscreening methods that are based upon whole cell assays, in vivoanalysis or transformed or immortal cell lines in which a reporter geneis employed to confer on its recombinant host(s) a readily detectablephenotype that emerges only under conditions where nor-1 and/or nur77nuclear receptor would have altered levels of its expression (such asincreased). As an example, reporter genes encode a polypeptide nototherwise produced by the host cell that is detectable by analysis,e.g., by chromogenic, fluorometric, radioisotopic or spectrophotometricanalysis. In a specific embodiment, at least part of nor-1 and/or nur77nuclear receptor polynucleotide that encodes the amino acid sequence hasbeen replaced with β-galactosidase, GFP, and the like.

[0074] Another example of a screening assay of the present invention ispresented herein. Nor-1 and/or nur77 nuclear receptor-expressing cellsare grown in microtiter wells, followed by addition of serial molarproportions of a candidate to a series of wells, and determination ofthe signal level after an incubation period that is sufficient todemonstrate expression in controls incubated solely with the vehiclethat was used to resuspend or dissolve the compound. The wellscontaining varying proportions of candidate are then evaluated forsignal activation. Candidates that demonstrate a dose-related increaseof reporter gene transcription or expression are then selected forfurther evaluation as clinical therapeutic agents for leukemia.

[0075] In an alternative embodiment there is a method for increasingnor-1 and/or nur77 nuclear receptor polynucleotide levels bytransfecting cells with nor-1 and/or nur77 nuclear receptorpolynucleotide. Delivery systems for tranfection of nucleic acids intocells may utilize either viral or non-viral methods. A skilled artisanrecognizes that a targeted system for non-viral forms of DNA or RNApreferably utilizes four components: 1) the DNA or RNA of interest; 2) amoiety that recognizes and binds to a cell surface receptor or antigen;3) a DNA binding moiety; and 4) a lytic moiety that enables thetransport of the complex from the cell surface to the cytoplasm.Further, liposomes and cationic lipids can be used to deliver thetherapeutic gene combinations to achieve the same effect. Potentialviral vectors include expression vectors derived from viruses such asadenovirus, vaccinia virus, herpes virus, and bovine papilloma virus. Inaddition, episomal vectors may be employed. Other DNA vectors andtransporter systems are known in the art.

[0076] One skilled in the art recognizes that expression vectors derivedfrom retroviruses, adenovirus, herpes or vaccinia viruses, or fromvarious bacterial plasmids, may be used for delivery of nucleotidessequences to a targeted organ, tissue or cell population. Methods whichare well known to those skilled in the art can be used to constructrecombinant vectors which will express nor-1 and/or nur77 nuclearreceptor polynucleotides.

[0077] In a specific embodiment, the transfection of nucleic acid isfacilitated by a transport protein, as described in Subramanian et al.(1999). Briefly, a peptide M9 is chemically bound to a cationic peptideas a carrier molecule. The cationic complex binds the negatively chargednucleic acid of interest, followed by binding of M9 to a nucleartransport protein, such as transportin.

[0078] In a specific embodiment, there is a method of treating anorganism with leukemia comprising administering therapeuticallyeffective levels to the organism of an amino acid or nucleic acidsequence of nor-1 and/or nur77 nuclear receptor.

[0079] In another embodiment, there is a method of preventing leukemiain an organism comprising the step of increasing levels of nor-1 and/ornur77 nuclear receptor nucleic acid or amino acid sequence. Theadministration can be to organisms that show no signs of the onset ofthe disease or have early signs of the disease. In a preferredembodiment, the organism is susceptible to the leukemia or shows agenetic predisposition to having leukemia.

[0080] In a preferred embodiment, the organism described herein to betreated or subject to preventative methods is a mammal, such as a human.

[0081] The methods and treatments described herein are directed toleukemia. In a specific embodiment, the disease is systemic, andtherapies would be administered to patients systemically. However, in analternative embodiment the therapies may be administered by directapplication, such as into the bone marrow.

[0082] Dosage and Formulation

[0083] The compounds (active ingredients) of this invention can beformulated and administered to treat leukemia by any means that producescontact of the active ingredient with the agent's site of action in thebody of a mammal. They can be administered by any conventional meansavailable for use in conjunction with pharmaceuticals, either asindividual therapeutic active ingredients or in a combination oftherapeutic active ingredients. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

[0084] The dosage administered will be a therapeutically effectiveamount of active ingredient and will, of course, vary depending uponknown factors such as the pharmacodynamic characteristics of theparticular active ingredient and its mode and route of administration;age, sex, health and weight of the recipient; nature and extent ofsymptoms; kind of concurrent treatment, frequency of treatment and theeffect desired.

[0085] The active ingredient can be administered orally in solid dosageforms such as capsules, tablets and powders, or in liquid dosage formssuch as elixirs, syrups, emulsions and suspensions. The activeingredient can also be formulated for administration parenterally byinjection, rapid infusion, nasopharyngeal absorption or dernoabsorption.The agent may be administered intramuscularly, intravenously,subcutaneously, transdermally or as a suppository. In administering acompound, the compound may be given systematically. For compounds whichrequire avoidance of systemic effects, a preferred embodiment isintrathecal administration. In a preferred embodiment, of the inventionthe compound is administered interarticularly for the treatment ofarthritis.

[0086] Gelatin capsules contain the active ingredient and powderedcarriers such as lactose, sucrose, mannitol, starch, cellulosederivatives, magnesium stearate, stearic acid, and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric coated forselective disintegration in the gastrointestinal tract.

[0087] Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

[0088] In general, water, a suitable oil, saline, aqueous dextrose(glucose), and related sugar solutions and glycols such as propyleneglycol or polyethylene glycols are suitable carriers for parenteralsolutions. Solutions for parenteral administration contain preferably awater-soluble salt of the active ingredient, suitable stabilizing agentsand, if necessary, buffer substances. Antioxidizing agents such assodium bisulfate, sodium sulfite or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium ethylenediaminetetraacetic acid (EDTA). Inaddition, parenteral solutions can contain preservatives such asbenzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, a standard reference text in this field.

[0089] Additionally, standard pharmaceutical methods can be employed tocontrol the duration of action. These are well known in the art andinclude control release preparations and can include appropriatemacromolecules, for example polymers, polyesters, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methyl cellulose,carboxymethyl cellulose or protamine sulfate. The concentration ofmacromolecules as well as the methods of incorporation can be adjustedin order to control release. Additionally, the agent can be incorporatedinto particles of polymeric materials such as polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers.In addition to being incorporated, these agents can also be used to trapthe compound in microcapsules.

[0090] Useful pharmaceutical dosage forms for administration of thecompounds of this invention can be illustrated as follows.Pharmacological ranges for the active ingredients can be determined bythe skilled artisan using methods well known in the art. Example rangesfor active ingredients are as follows: folate ranges between 400micrograms and 4 milligrams/day; methionine ranges between 250 mg(total)and as high as 100 mg/kg/day daily, up to 2-3 g; choline ranges between100 mg and 2 grams; Vitamin B12 at approximately 100 micrograms orallyor 1 mg intramuscularly per month; betaine ranges up to 6 grams per day;zinc ranges between 25 and 50 mg; and sodium phenylbutyrate ranges up to20 grams per day.

[0091] Capsules: Capsules are prepared by filling standard two-piecehard gelatin capsulates each with powdered active ingredient, 175milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesiumstearate.

[0092] Soft Gelatin Capsules: A mixture of active ingredient in soybeanoil is prepared and injected by means of a positive displacement pumpinto gelatin to form soft gelatin capsules containing the activeingredient. The capsules are then washed and dried.

[0093] Tablets: Tablets are prepared by conventional procedures so thatthe dosage unit contains the suggested amount of active ingredient, 0.2milligrams of colloidal silicon dioxide, 5 milligrams of magnesiumstearate, 275 milligrams of microcrystalline cellulose, 11 milligrams ofcornstarch and 98.8 milligrams of lactose. Appropriate coatings may beapplied to increase palatability or to delay absorption.

[0094] Injectable: A parenteral composition suitable for administrationby injection is prepared by stirring 1.5% by weight of activeingredients in 10% by volume propylene glycol and water. The solution ismade isotonic with sodium chloride and sterilized.

[0095] Suspension: An aqueous suspension is prepared for oraladministration so that each 5 milliliters contains the suggested amountof finely divided active ingredient, 200 milligrams of sodiumcarboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams ofsorbitol solution U.S. Pat. No. and 0.025 milliliters of vanillin.

[0096] Accordingly, the pharmaceutical composition of the presentinvention may be delivered via various routes and to various sites in ananimal body to achieve a particular effect. One skilled in the art willrecognize that although more than one route can be used foradministration, a particular route can provide a more immediate and moreeffective reaction than another route. Local or systemic delivery can beaccomplished by administration comprising application or instillation ofthe formulation into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, peritoneal, subcutaneous, intradermal, as well as topicaladministration.

[0097] The composition of the present invention can be provided in unitdosage form wherein each dosage unit, e.g., a teaspoonful, tablet,solution, or suppository, contains a predetermined amount of thecomposition, alone or in appropriate combination with other activeagents. The term “unit dosage form” as used herein refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of thecompositions of the present invention, alone or in combination withother active agents, calculated in an amount sufficient to produce thedesired effect, in association with a pharmaceutically acceptablediluent, carrier, or vehicle, where appropriate. The specifications forthe unit dosage forms of the present invention depend on the particulareffect to be achieved and the particular pharmacodynamics associatedwith the pharmaceutical composition in the particular host.

[0098] These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

[0099] In a specific embodiment, a drug may be transported to a targetby utilizing carbonic anhydrase inhibitor (CAI) which contains a polargroup such as a carboxyl group, as described in Kehayova et al., 1999.The carboxyl group renders the composition dissolvable in water,however, upon exposure to light the bond linking the CAI to the carboxylmask breaks, allowing the remaining portion to be soluble in ahydrophobic environment.

[0100] In certain embodiments, the use of lipid formulations and/ornanocapsules is contemplated for the introduction of, for example, anagonist to nor-1 and/or nur77 nuclear receptor, a polypeptide comprisingnor-1 and/or nur77 nuclear receptor amino acid sequence, a nucleic acidcomprising nor-1 and/or nur77 nuclear receptor, or pharmaceuticallyacceptable salts thereof, polypeptides, peptides and/or agents, and/orgene therapy vectors, including both wild-type and/or antisense vectors,into host cells.

[0101] Nanocapsules can generally entrap compounds in a stable and/orreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and/or such particles maybe easily made.

[0102] In a preferred embodiment, of the invention, the pharmaceuticalcomposition may be associated with a lipid. The pharmaceuticalcomposition associated with a lipid may be encapsulated in the aqueousinterior of a liposome, interspersed within the lipid bilayer of aliposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the oligonucleotide, entrapped ina liposome, complexed with a liposome, dispersed in a solutioncontaining a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, orotherwise associated with a lipid. The lipid or lipid/pharmaceuticalcomposition associated compositions of the present invention are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in either size or shape.

[0103] Lipids are fatty substances that may be naturally occurring orsynthetic lipids. For example, lipids include the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds thatare well-known to those of skill in the art which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

[0104] Phospholipids may be used for preparing the liposomes accordingto the present invention and may carry a net positive, negative, orneutral charge. Diacetyl phosphate can be employed to confer a negativecharge on the liposomes, and stearylamine can be used to confer apositive charge on the liposomes. The liposomes can be made of one ormore phospholipids.

[0105] A neutrally charged lipid can comprise a lipid with no charge, asubstantially uncharged lipid, or a lipid mixture with equal number ofpositive and negative charges. Suitable phospholipids includephosphatidyl cholines and others that are well known to those of skillin the art.

[0106] Lipids suitable for use according to the present invention can beobtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma Chemical Co.,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Chol”) is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Preferably, chloroform is used as theonly solvent since it is more readily evaporated than methanol.

[0107] Phospholipids from natural sources, such as egg or soybeanphosphatidylcholine, brain phosphatidic acid, brain or plantphosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine are preferably not used as the primaryphosphatide, i.e., constituting 50% or more of the total phosphatidecomposition, because of the instability and leakiness of the resultingliposomes.

[0108] “Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

[0109] Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strengthand/or the presence of divalent cations. Liposomes can show lowpermeability to ionic and/or polar substances, but at elevatedtemperatures undergo a phase transition which markedly alters theirpermeability. The phase transition involves a change from a closelypacked, ordered structure, known as the gel state, to a loosely packed,less-ordered structure, known as the fluid state. This occurs at acharacteristic phase-transition temperature and/or results in anincrease in permeability to ions, sugars and/or drugs.

[0110] Liposomes interact with cells via four different mechanisms:Endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and/or neutrophils; adsorption to the cell surface,either by nonspecific weak hydrophobic and/or electrostatic forces,and/or by specific interactions with cell-surface components; fusionwith the plasma cell membrane by insertion of the lipid bilayer of theliposome into the plasma membrane, with simultaneous release ofliposomal contents into the cytoplasm; and/or by transfer of liposomallipids to cellular and/or subcellular membranes, and/or vice versa,without any association of the liposome contents. Varying the liposomeformulation can alter which mechanism is operative, although more thanone may operate at the same time.

[0111] Liposome-mediated oligonucleotide delivery and expression offoreign DNA in vitro has been very successful. Wong et al. (1980)demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa and hepatomacells. Nicolau et al. (1987) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection.

[0112] In certain embodiments of the invention, the lipid may beassociated with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the lipid may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the lipid may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression vectorshave been successfully employed in transfer and expression of anoligonucleotide in vitro and in vivo, then they are applicable for thepresent invention. Where a bacterial promoter is employed in the DNAconstruct, it also will be desirable to include within the liposome anappropriate bacterial polymerase.

[0113] Liposomes used according to the present invention can be made bydifferent methods. The size of the liposomes varies depending on themethod of synthesis. A liposome suspended in an aqueous solution isgenerally in the shape of a spherical vesicle, having one or moreconcentric layers of lipid bilayer molecules. Each layer consists of aparallel array of molecules represented by the formula XY, wherein X isa hydrophilic moiety and Y is a hydrophobic moiety. In aqueoussuspension, the concentric layers are arranged such that the hydrophilicmoieties tend to remain in contact with an aqueous phase and thehydrophobic regions tend to self-associate. For example, when aqueousphases are present both within and without the liposome, the lipidmolecules may form a bilayer, known as a lamella, of the arrangementXY-YX. Aggregates of lipids may form when the hydrophilic andhydrophobic parts of more than one lipid molecule become associated witheach other. The size and shape of these aggregates will depend upon manydifferent variables, such as the nature of the solvent and the presenceof other compounds in the solution.

[0114] Liposomes within the scope of the present invention can beprepared in accordance with known laboratory techniques. In onepreferred embodiment, liposomes are prepared by mixing liposomal lipids,in a solvent in a container, e.g., a glass, pear-shaped flask. Thecontainer should have a volume ten-times greater than the volume of theexpected suspension of liposomes. Using a rotary evaporator, the solventis removed at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

[0115] Dried lipids can be hydrated at approximately 25-50 mMphospholipid in sterile, pyrogen-free water by shaking until all thelipid film is resuspended. The aqueous liposomes can be then separatedinto aliquots, each placed in a vial, lyophilized and sealed undervacuum.

[0116] In the alternative, liposomes can be prepared in accordance withother known laboratory procedures: the method of Bangham et al. (1965),the contents of which are incorporated herein by reference; the methodof Gregoriadis, as described in DRUG CARRIERS IN BIOLOGY AND MEDICINE,G. Gregoriadis ed. (1979) pp. 287-341, the contents of which areincorporated herein by reference; the method of Deamer and Uster (1983),the contents of which are incorporated by reference; and thereverse-phase evaporation method as described by Szoka andPapahadjopoulos (1978). The aforementioned methods differ in theirrespective abilities to entrap aqueous material and their respectiveaqueous space-to-lipid ratios.

[0117] The dried lipids or lyophilized liposomes prepared as describedabove may be dehydrated and reconstituted in a solution of inhibitorypeptide and diluted to an appropriate concentration with an suitablesolvent, e.g., DPBS. The mixture is then vigorously shaken in a vortexmixer. Unencapsulated nucleic acid is removed by centrifugation at29,000×g and the liposomal pellets washed. The washed liposomes areresuspended at an appropriate total phospholipid concentration, e.g.,about 50-200 mM. The amount of nucleic acid encapsulated can bedetermined in accordance with standard methods. After determination ofthe amount of nucleic acid encapsulated in the liposome preparation, theliposomes may be diluted to appropriate concentrations and stored at 4°C. until use.

[0118] A pharmaceutical composition comprising the liposomes willusually include a sterile, pharmaceutically acceptable carrier ordiluent, such as water or saline solution.

[0119] Gene Therapy Administration

[0120] For gene therapy, a skilled artisan would be cognizant that thevector to be utilized must contain the gene of interest operativelylinked to a promoter. One skilled in the art recognizes that in certaininstances other sequences such as a 3′ UTR regulatory sequences areuseful in expressing the gene of interest. Where appropriate, the genetherapy vectors can be formulated into preparations in solid, semisolid,liquid or gaseous forms in the ways known in the art for theirrespective route of administration. Means known in the art can beutilized to prevent release and absorption of the composition until itreaches the target organ or to ensure timed-release of the composition.A pharmaceutically acceptable form should be employed which does notineffectuate the compositions of the present invention. Inpharmaceutical dosage forms, the compositions can be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds. A sufficient amount of vectorcontaining the therapeutic nucleic acid sequence must be administered toprovide a pharmacologically effective dose of the gene product.

[0121] One skilled in the art recognizes that different methods ofdelivery may be utilized to administer a vector into a cell. Examplesinclude: (1) methods utilizing physical means, such as electroporation(electricity), a gene gun (physical force) or applying large volumes ofa liquid (pressure); and (2) methods wherein the vector is complexed toanother entity, such as a liposome or transporter molecule.

[0122] Accordingly, the present invention provides a method oftransferring a therapeutic gene to a host, which comprises administeringthe vector of the present invention, preferably as part of acomposition, using any of the aforementioned routes of administration oralternative routes known to those skilled in the art and appropriate fora particular application. Effective gene transfer of a vector to a hostcell in accordance with the present invention to a host cell can bemonitored in terms of a therapeutic effect (e.g. alleviation of somesymptom associated with the particular disease being treated) or,further, by evidence of the transferred gene or expression of the genewithin the host (e.g., using the polymerase chain reaction inconjunction with sequencing, Northern or Southern hybridizations, ortranscription assays to detect the nucleic acid in host cells, or usingimmunoblot analysis, antibody-mediated detection, mRNA or proteinhalf-life studies, or particularized assays to detect protein orpolypeptide encoded by the transferred nucleic acid, or impacted inlevel or function due to such transfer).

[0123] These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

[0124] Furthermore, the actual dose and schedule can vary depending onwhether the compositions are administered in combination with otherpharmaceutical compositions, or depending on interindividual differencesin pharmacokinetics, drug disposition, and metabolism. Similarly,amounts can vary in in vitro applications depending on the particularcell line utilized (e.g., based on the number of vector receptorspresent on the cell surface, or the ability of the particular vectoremployed for gene transfer to replicate in that cell line). Furthermore,the amount of vector to be added per cell will likely vary with thelength and stability of the therapeutic gene inserted in the vector, aswell as also the nature of the sequence, and is particularly a parameterwhich needs to be determined empirically, and can be altered due tofactors not inherent to the methods of the present invention (forinstance, the cost associated with synthesis). One skilled in the artcan easily make any necessary adjustments in accordance with theexigencies of the particular situation.

[0125] It is possible that cells containing the therapeutic gene mayalso contain a suicide gene (i.e., a gene which encodes a product thatcan be used to destroy the cell, such as herpes simplex virus thymidinekinase). In many gene therapy situations, it is desirable to be able toexpress a gene for therapeutic purposes in a host cell but also to havethe capacity to destroy the host cell once the therapy is completed,becomes uncontrollable, or does not lead to a predictable or desirableresult. Thus, expression of the therapeutic gene in a host cell can bedriven by a promoter although the product of the suicide gene remainsharmless in the absence of a prodrug. Once the therapy is complete or nolonger desired or needed, administration of a prodrug causes the suicidegene product to become lethal to the cell. Examples of suicidegene/prodrug combinations which may be used are Herpes SimplexVirus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU;oxidoreductase and cycloheximide; cytosine deaminase and5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) andAZT; and deoxycytidine kinase and cytosine arabinoside.

[0126] The method of cell therapy may be employed by methods known inthe art wherein a cultured cell containing a non-defective nor-1 and/ornur77 nuclear receptor nucleic acid sequence encoding nor-1 and/or nur77nuclear receptor polypeptide is introduced.

[0127] In another embodiment, biologically active molecules, such asvectors for gene therapy, are incorporated in a large hydration domainbetween “pinched” regions of a lipid-poly-L-glutamic acid (PGA) complex,where the PGA and the cationic lipid didodecyl dimethylammonium bromideassociate to form localized pinched regions, for delivery applications(Subramaniam, et al., 2000).

[0128] In an alternative embodiment, an amino acid sequence isengineered to accumulate as an aggregate in the endoplasmic reticulum,followed by administration of a composition to induce proteindisaggregation, resulting in rapid and transient secretion (Rivera etal., 2000).

[0129] A peptide (11 amino acids) derived from HIV has been recentlydescribed that when fused to full length proteins and injected into miceallow a rapid dispersal to the nucleus of all cells of the body(Schwarze et al., 1999). Schwarze et al. made fusion proteins to Tatranging in size from 15 to 120 kDa. They documented a rapid uptake ofthe fusion proteins to the nuclei of cells throughout the animal, andthe functional activity of the proteins was retained.

[0130] In an embodiment of the present invention there are constructscontaining the Tat or Tat-HA nucleic acid sequence operatively linked tothe nor-1 and/or nur77 nuclear receptor nucleic acid sequence. Thevectors are expressed in bacterial cultures and the fusion protein ispurified. This purified Tat-HA-nor-1/nur77 nuclear receptor protein orTat-nor-1/nur77 nuclear receptor protein is injected into the animal todetermine the efficiency of the Tat delivery system into the particularsite of delivery, such as into the bone marrow, or by means to deliverthe fusion protein systemically. Analysis is carried out to determinethe potential of the Tat-HA-nor-1/nur77 nuclear receptor protein orTat-nor-1/nur77 nuclear receptor protein in alleviation of any leukemiasymptom. This is a viable therapeutic approach either in its own rightor in association with other methods, treatments or genes.

[0131] DNA Delivery Using Viral Vectors

[0132] The ability of certain viruses to infect cells viareceptor-mediated endocytosis, to integrate into host cell genome and toexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells.Preferred gene therapy vectors of the present invention will generallybe viral vectors.

[0133] Although some viruses that can accept foreign genetic materialare limited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

[0134] Of course, in using viral delivery systems, one will desire topurify the virion sufficiently to render it essentially free ofundesirable contaminants, such as defective interfering viral particles,endotoxins, and other pyrogens such that it will not cause any untowardreactions in the cell, animal or individual receiving the vectorconstruct. A preferred means of purifying the vector involves the use ofbuoyant density gradients, such as cesium chloride gradientcentrifugation.

[0135] a. Adenoviral Vectors

[0136] A particular method for delivery of the expression constructsinvolves the use of an adenovirus expression vector. Although adenovirusvectors are known to have a low capacity for integration into genomicDNA, this feature is counterbalanced by the high efficiency of genetransfer afforded by these vectors. “Adenovirus vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to ultimately express atissue or cell-specific construct that has been cloned therein.

[0137] The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhausand/or Horwitz, 1992). In contrast to retrovirus, the adenoviralinfection of host cells does not result in chromosomal integrationbecause adenoviral DNA can replicate in an episomal manner withoutpotential genotoxicity. Also, adenoviruses are structurally stable, andno genome rearrangement has been detected after extensive amplification.

[0138] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0139] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0140] Generation and/or propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from embryonic kidney cells by Ad5DNA fragments and constitutively expresses E1 proteins (E1A and/orGraham et al., 1977). Since the E3 region is dispensable from theadenovirus genome (Jones and Shenk, 1978), the current adenovirusvectors, with the help of 293 cells, carry foreign DNA in either the E1,the D3 or both regions (Graham and Prevec, 1991). Recently, adnoviralvectors comprising deletions in the E4 region have been described (U.S.Pat. No. 5,670,488, incorporated herein by reference).

[0141] In nature, adenovirus can package approximately 105% of thewild-type genome (Ghosh-Choudhury et al., 1987), providing capacity forabout 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNAthat is replaceable in the E1 and/or E3 regions, the maximum capacity ofthe current adenovirus vector is under 7.5 kb, and/or about 15% of thetotal length of the vector. More than 80% of the adenovirus viral genomeremains in the vector backbone.

[0142] Helper cell lines may be derived from mammalian cells such ashuman embryonic kidney cells, muscle cells, hematopoietic cells andother human embryonic mesenchymal or epithelial cells. Alternatively,the helper cells may be derived from the cells of other mammalianspecies that are permissive for adenovirus. Such cells include, e.g.,Vero cells and/or other monkey embryonic mesenchymal and/or epithelialcells. As stated above, the preferred helper cell line is 293.

[0143] Recently, Racher et al. (1995) disclosed improved methods forpropagating adenovirus. In one format, natural cell aggregates are grownby inoculating individual cells into 1 liter siliconized spinner flasks(Techne, Cambridge, UK) containing 100-200 ml of medium. Followingstirring at 40 rpm, the cell viability is estimated with trypan blue. Inanother format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5g/l) is employed as follows. A cell inoculum, resuspended in 5 ml ofmedium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flaskand/or left stationary, with occasional agitation, for 1 to 4 h. Themedium is then replaced with 50 ml of fresh medium and/or shakinginitiated. For virus production, cells are allowed to grow to about 80%confluence, after which time the medium is replaced (to 25% of the finalvolume) and/or adenovirus added at an MOI of 0.05. Cultures are leftstationary overnight, following which the volume is increased to 100%and/or shaking commenced for another 72 h.

[0144] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes and subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a adenovirusabout which a great deal of biochemical and genetic information isknown, and it has historically been used for most constructionsemploying adenovirus as a vector.

[0145] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the transformingconstruct at the position from which the E1-coding sequences have beenremoved. However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the NURR subfamily member may also be insertedin lieu of the deleted E3 region in E3 replacement vectors as describedby Karlsson et al (1986) or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

[0146] Adenovirus growth and manipulation is known to those of skill inthe art, and exhibits broad host range in vitro and in vivo. This groupof viruses can be obtained in high titers, e.g., 10⁹ to 10¹¹plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. No side effectshave been reported in studies of vaccination with wild-type adenovirus(Couch et al., 1963; Top et al, 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

[0147] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and/or Horwitz, 1992; Graham and/or Prevec, 1992). Recently,animal studies suggested that recombinant adenovirus could be used forgene therapy (Stratford-Perricaudet and/or Perricaudet, 1991a;Stratford-Perricaudet et al., 1991b; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al, 1993), peripheral intravenous injections(Herz and/or Gerard, 1993) and stereotactic inoculation into the brain(Le Gal La Salle et al., 1993). Recombinant adenovirus andadeno-associated virus (see below) can both infect and transducenon-dividing mammalian primary cells.

[0148] b. Adeno-Associated Viral Vectors

[0149] Adeno-associated virus (AAV) is an attractive vector system foruse in the cell transduction of the present invention as it has a highfrequency of integration, and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) and in vivo. AAV has a broadhost range for infectivity (Tratschin et al., 1984; Laughlin et al.,1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated hereinby reference.

[0150] Studies demonstrating the use of AAV in gene delivery includeLaFace et al. (1988); Zhou et al. (1993); Flotte et al. (1993); andWalsh et al. (1994). Recombinant AAV vectors have been used successfullyfor in vitro and in vivo transduction of marker genes (Kaplitt et al.,1994; Lebkowski et al., 1988; Samulski et al., 1989; Yoder et al., 1994;Zhou et al., 1994; Hermonat and/or Muzyczka, 1984; Tratschin et al.,1985; McLaughlin et al., 1988) or genes involved in mammalian diseases(Flotte et al., 1992; Luo et al., 1994; Ohi et al., 1990; Walsh et al.,1994; Wei et al., 1994). Recently, an AAV vector has been approved forphase I trials for the treatment of cystic fibrosis.

[0151] AAV is a dependent parvovirus in that it requires coinfectionwith another virus (either adenovirus or a member of the herpes virusfamily) to undergo a productive infection in cultured cells (Muzyczka,1992). In the absence of coinfection with helper virus, the wild typeAAV genome integrates through its ends into chromosome 19 where itresides in a latent state as a provirus (Kotin et al., 1990; Samulski etal., 1991). rAAV, however, is not restricted to chromosome 19 forintegration unless the AAV Rep protein is also expressed (Shelling andSmith, 1994). When a cell carrying an AAV provirus is superinfected witha helper virus, the AAV genome is “rescued” from the chromosome or froma recombinant plasmid, and a normal productive infection is established(Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990;Muzyczka, 1992).

[0152] Typically, recombinant AAV (rAAV) virus is made by cotransfectinga plasmid containing the gene of interest flanked by the two AAVterminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; eachincorporated herein by reference) and an expression plasmid containingthe wild type AAV coding sequences without the terminal repeats, forexample pEM45 (McCarty et al., 1991; incorporated herein by reference).The cells are also transfected with adenovirus or plasmids carrying theadenovirus genes required for AAV helper function. rAAV virus stocksmade in such fashion are contaminated with adenovirus which must bephysically separated from the rAAV particles (for example, by cesiumchloride density centrifugation). Alternatively, adenovirus vectorscontaining the AAV coding regions or cell lines containing the AAVcoding regions and some or all of the adenovirus helper genes could beused (Yang et al., 1994; Clark et al., 1995). Cell lines carrying therAAV DNA as an integrated provirus can also be used (Flotte et al.,1995).

[0153] C. Retroviral Vectors

[0154] Retroviruses have promise as gene delivery vectors due to theirability to integrate their genes into the host genome, transferring alarge amount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines(Miller, 1992).

[0155] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0156] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolasand/or Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

[0157] Concern with the use of defective retrovirus vectors is thepotential appearance of wild-type replication-competent virus in thepackaging cells. This can result from recombination events in which theintact sequence from the recombinant virus inserts upstream from thegag, pol, and env sequences integrated in the host cell genome. However,new packaging cell lines are now available that should greatly decreasethe likelihood of recombination (Markowitz et al, 1988; Hersdorffer etal., 1990).

[0158] Gene delivery using second generation retroviral vectors has beenreported. Kasahara et al. (1994) prepared an engineered variant of theMoloney murine leukemia virus, which normally infects only mouse cells,that modified an envelope protein so that the virus specifically boundto, and infected, mammalian cells bearing the erythropoietin (EPO)receptor. This was achieved by inserting a portion of the EPO sequenceinto an envelope protein to create a chimeric protein with a new bindingspecificity.

[0159] d. Other Viral Vectors

[0160] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Baichwal and/or Sugden, 1986; Coupar et al.,1988), sindbis virus, cytomegalovirus and herpes simplex virus may beemployed. They offer several attractive features for various mammaliancells (Friedmann, 1989; Ridgeway, 1988; Baichwal and/or Sugden, 1986;Coupar et al., 1988; Horwich et al., 1990).

[0161] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

[0162] In certain further embodiments, the gene therapy vector will beHSV. A factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (temporal, strength, etc.)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and can be grown to high titers. Thus,delivery is less of a problem, both in terms of volumes needed to attainsufficient MOI and in a lessened need for repeat dosings.

[0163] e. Modified Viruses

[0164] In still further embodiments of the present invention, thenucleic acids to be delivered are housed within an infective virus thathas been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was recentlydeveloped based on the chemical modification of a retrovirus by thechemical addition of lactose residues to the viral envelope. Thismodification can permit the specific infection of hepatocytes viasialoglycoprotein receptors.

[0165] Another approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein or against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of mammalian cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

EXAMPLES

[0166] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1

[0167] Pathology of Mice Deficient for Nor-1 and Nur77

[0168] Mice deficient for both nor-1 and nur77 (nor-1KO/nur77KO) weregenerated. Shortly after birth, the nor-1KO/nur77KO mice begin to wasteand appear lethargic. FIG. 1 shows the growth curve of one litterincluding two nor-1KO/nur77KO mice and their normal littermates that isrepresentative of a number of litters that were weighed daily for aperiod of two weeks. After a short period of weight loss and increasingweakness, these mice become moribund and succumb to death. The postnatalday of death varies greatly in these mice, however, generally occursprior to the fourth week of life. Upon necropsy the nor-1KO/nur77KO micedisplay lymphadenopathy and splenomegaly (FIG. 2). The lymph node andspleen are both lymphoid tissues and these defects suggested alterationin hematopoiesis. In addition, liver discoloration consistent withcellular infiltration was noted (FIG. 3).

[0169] Blood samples were analyzed from several nor-1KO/nur77KO miceshowing disease symptoms. Table 1 shows the results obtained from twonor-1KO/nur77KO mice at varying ages and their corresponding normallittermates. Both of the double knockout mice showed elevated totalwhite blood cells (leukocytosis). TABLE 1 Peripheral Blood Counts atPostnatal Day 14-16 nor-1/nur77 WT/WT KO/KO WBC × 2.86 ± 0.5 7.70 ±0.7^(c) 10 {circumflex over ( )} 3/uL^(a) RBC × 5.43 ± 0.3 3.23 ±0.5^(c) 10 {circumflex over ( )} 6/uL^(a) HGB g/dL^(a) 10.7 ± 0.6 6.17 ±1.3^(c) % HCT^(a) 32.5 ± 1.2 19.5 ± 3.2^(c) % Neutrophils^(b) 23.0 ± 2.014.0 ± 5.0  % Lymphocytes^(b) 70.0 ± 2.0 13.0 ± 7.0^(c) % Monocytes^(b) 0 ± 0  0 ± 0  % Eosinophils^(b)  4.0 ± 1.0  0 ± 0^(c) % Basophils^(b) 0 ± 0  0 ± 0   % Young Forms^(b)  3.0 ± 1.0 84.0 ± 4.0^(c)

[0170] Automated and manual differential analysis of the peripheralblood in the nor-1KO/nur77KO mice compared to the normal littermatesshowed a substantial increase in the percentage and total number ofneutrophils (neutrophilia) in double knock out mice. The double knockout mice were anemic, illustrated by pale blood color, decreased numberof red blood cells and decreased concentration of hemoglobin, and lowerhematocrit values. Leukocytosis, neutrophilia, and anemia are classicsymptoms of leukemia in both human patients and other mouse models ofleukemia. An increase in young myeloid forms was also noted duringdifferential analysis and is often observed in patients with variousforms of myeloid leukemia.

[0171] Histological examination of lymphoid tissues of thenor-1KO/nur77KO animals showed disrupted architecture (FIG. 4).Specifically, the spleen showed loss of distinct lymphocytic nodulesthat is seen in normal spleens. Normally, the thymus contains a distinctdarkly staining cortex and a paler staining medulla. However thenor1KO/nur77KO thymus has lost the classic cortical-medullaryarchitecture. In addition, abnormal encapsulation and tumor-like septaewere present in the thymus. The pale staining appearance of the thymussuggested to us that medullary epithelial cells might be part of thisabnormal histology. Immunohistochemistry using antibodies specific tomedullary epithelial cells confirmed this suspicion (FIG. 5). Disruptedarchitecture in both the spleen and the thymus is consistent with aglobal hematopoietic disorder such as leukemia.

[0172] Based on previous results implicationg both nor-1 and nur77during negative selection in the thymus, it was possible thehistological defects mentioned above were due to abnormal survival oflymphocytes that would normally die during negative selection.Thymocytes were harvested from mice at postnatal day 7 and counted on ahematocytometer. In contrast to the predictions, consistently lessthymocytes were obtained from the nor-1KO/nur77KO thymuses as comparedto normal thymuses (FIG. 6). Even more unexpected, flow cytometricanalysis of well defined developmental stages based on expression ofcell surface antigens CD4 and CD8 showed that this decrease was notlimited to the later stage of negative selection (FIG. 7). These datashow that nor-1 and nur77 play a role in early lymphocyte developmentprior to negative selection within the thymus and/or the bone marrowthat has never been reported before.

[0173] Histology of nonlymphoid tissues obtained from nor-1KO/nur77KOmice revealed extensive perivascular cellular infiltration in the lung,liver, and pancreas, tissues that are often affected in leukemicpatients (FIG. 8). These infiltrates within the lung were extensiveenough to speculate that the opening of the airways of these mice may beimpeded which could explain the labored breathing observed in themoribund mice just prior to death.

[0174] To further characterize the disrupted histology within thenor-1KO/nur77KO mice, flow cytometry was used to analyze the cell typeswithin the lymphoid tissues and peripheral blood. Analysis of CD11b, acell surface marker for the myeloid lineage and GR-1 a marker forgranulocytes that differentiate from the myeloid lineage revealed andincrease of CD11b+ and CD11b+/GR+ cells within the thymus, peripheralblood, spleen, and lymph node of diseased animals as compared to normallittermates (FIG. 9). This data is consistent with the neutrophiliafound during blood analysis of the nor-1KO/nur77KO mice. CD11b and GR-1expression are increased in blood from patients with both chronic andacute myeloid leukemia.

[0175] The flow cytometry data showing increased levels of CD11b+ cellsand neutrophilia detected in blood analysis from the knock out micesuggested that the abnormal cellular infiltrates observed in thenonlymphoid tissues might consist of myeloid cells. This was confirmedby staining histological tissue sections of the cellular infiltrateswith myeloperoxidase, a stain often used to identify cells of myeloidorigin in tissue samples and blood obtained from patients with myeloidleukemia (FIG. 10). The presence of leukemic myeloid cells within boththe lymphoid and nonlymphoid tissues was further confirmed withimmunohistochemistry using an antibody that binds to the CD11b moleculeon the cell surfaces (FIG. 11).

[0176] The above data lead to the diagnosis of myeloid leukemia withdifferentiation similar to that of human chronic myeloid leukemia indiseased nor-1KO/nur77KO mice. Leukemia is a result of a primary defectof hematopoiesis within the bone marrow. Therefore, the bone marrow ofnor-1KO/nur77KO mice was examined using flow cytometry. It has beenreported that while leukemia begins within the bone marrow, changes incell composition sometimes aren't detected due to the rapid exit of theabnormal cells into the periphery although this is not always the casein well developed leukemia. The analysis revealed an increase in thepercentage of CD11b+ myeloid cells (FIG. 12). In addition, decreasedpercentages of B220+ cells of the b-lymphocyte population were observed.A shift in hematopoiesis to production of CD11b+ cells and a decrease inother hematopoietic lineages is consistent with what is observed in bothacute and chronic leukemia. These results also confirm and essentialrole of nor-1 and nur77 in regulation of bone marrow hematopoiesis andprevention of the development of leukemia.

Example 2

[0177] Pathology of Hypoallelic Nor-1 and Nur77 Deficient Mice

[0178] While breeding to obtain nor-1KO/nur77KO mice, mice with oneallele of either gene remaining, nor-1±/nur77KO or nor-1KO/nur77± mice,were generated. These mice approximately contain one quarter of thenormal level of total nor-1 and nur77 protein. These mice are referredto herein as hypoallelic.

[0179] Initially these mice appear normal. However, by 3-4 months ofage, they begin to show similar outward signs of disease as thenor-1KO/nur77KO mice. Upon necropsy of the diseased animals,splenomegaly, lymphadenopathy, and discoloration of the liver was noted.Histological examination of the lymphoid tissue revealed a similarphenotype as the nor-1KO/nur77KO (FIG. 13). The normalcortical/medullary junction of the thymus has been lost in thesehypoallelic mice. The spleen of these mice has lost distinct nodulararchitecture. In addition, like the nor-1KO/nur77KO mice, extensiveperivascular cellular infiltrates were noted in the liver, lung, andpancreas (FIG. 14). To further confirm that the hypoallelic mice werealso developing myeloid leukemia, blood analysis was performed (Table2). TABLE 2 Peripheral Blood Analysis of Hypoallelic mice in comparisonto normal littermate. AGE (months) WBC × 10³/ul RBC × 10⁶/ul HGB g/dLHCT % % Neutrophils % LUC KO/HE 5 4.75 7.24 11.7 33 61.1 2 HE/KO 3 6.59.22 13.7 43.5 23.5 2.7 WT/KO 3 1.25 9.2 14.5 45.5 8.8 0.9

[0180] Just as in the nor-1KO/nur77KO animals, the hypoallelic animalsshowed leukocytosis, neutrophilia, anemia, and an increase in thepercentage of large unstained cells as compared to normal levels. Largeunstained cells are often increased in automated count values when younghematopoietic cells are present in the circulation. The hypoallelic micealso develop myeloid leukemia, however, at a later age. This differenceshows that even at one-quarter of their normal level and activity, nor-1or nur77 can maintain regulated myeloid hematopoiesis and prevent theonset of leukemia for a period of time. Thus, the level of expression oractivity of nor-1 or nur77 is critical to protection against thedevelopment of leukemia.

[0181] Thus, rapidly developing myeloid leukemia in the absence of nor-1and nur77 show that these genes are critical during hematopoiesis. Noreports have ever implicated nor-1, nur77, or their relative nurr1during this process. In addition, the hypoallelic mouse model providedherein that retains only one-quarter of the normal level and activity ofnor-1 or nur77 also develops leukemia, although at a later stage. Thisillustrates how crucial the amount and activity of these nuclearreceptors is during hematopoiesis. In specific embodiments, increasingeither the level or activity of nor-1 or nur77 prevents against theonset of or alters the progression of unregulated proliferation ofhematopoietic cells that occurs during myeloid leukemia.

[0182] References

[0183] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0184] Patents

[0185] U.S. Pat. No. 4,797,368

[0186] U.S. Pat. No. 5,139,941

[0187] U.S. Pat. No. 5,670,488

[0188] U.S. Pat. No. 6,541,217

[0189] Publications

[0190] Bandoh, S. et al. Mechanical agitation induces gene expression ofNOR-1 and its closely related orphan nuclear receptors in leukemic celllines. Leukemia (1997) 11:1453-1458.

[0191] Chang, P. K., Cary, J. W., Bhatnagar, D., Cleveland, T. E.,Bennett, J. W., Linz, J. E., Woloshuk, C. P. and Payne, G. A. Cloning ofthe Aspergillus parasiticus apa-2 gene associated with the regulation ofaflatoxin biosynthesis. Appl. Environ. Microbiol. 59 (10), 3273-3279(1993).

[0192] Wu W. -S. et al. Promyelocytic leukemia protein PML inhibitsNur77-mediated transcription through specific functional interactions.Oncogene (2002) 21: 3925-3933.

[0193] Although the present invention and its advantages have beendescribed in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe spirit and scope of the invention as defined by the appended claims.Moreover, the scope of the present application is not intended to belimited to the particular embodiments of the process, machine,manufacture, composition of matter, means, methods and steps describedin the specification. As one of ordinary skill in the art will readilyappreciate from the disclosure of the present invention, processes,machines, manufacture, compositions of matter, means, methods, or steps,presently existing or later to be developed that perform substantiallythe same function or achieve substantially the same result as thecorresponding embodiments described herein may be utilized according tothe present invention. Accordingly, the appended claims are intended toinclude within their scope such processes, machines, manufacture,compositions of matter, means, methods, or steps.

1 36 1 3802 DNA Homo sapiens 1 ataaatgacg tgccgagaga gcgagcgaacgcgcagccgg gagagcggag tctcctgcct 60 cccgcccccc acccctccag ctcctgctcctcctccgctc cccatacaca gacgcgctca 120 cacccgctcc ctcactcgaa cacacagacacaagcgcgca cacaggctcc gcacacacac 180 acttcgctct cccgcgcgct cacacccctcttgccctgag cccttgccgg tgcagcgcgg 240 cgccgcagct ggacgcccct cccgggctcactttgcaacg ctgacggtgc cggcagtggc 300 cgtggaggtg ggaacagcgg cggcatcctcccccctggtc acagcccaag ccaggacgcc 360 cgcggaacct ctcggctgtg ctctcccatgagtcgggatc gcagcatccc ccaccagccg 420 ctcaccgcct ccgggagccg ctgggcttgtacaccgcagc ccttccggga cagcagctgt 480 gactcccccc cagtgcagat ttcgggacagctctctagaa actcgctcta aagacggaac 540 cgccacagca ctcaaagccc actgcggaagagggcagccc ggcaagcccg ggccctgagc 600 ctggaccctt agcggtgccg ggcagcactgccggcgcttc gcctcgccgg acgtccgctc 660 ctcctacact ctcagcctcc gctggagagacccccagccc caccattcag cgcgcaagat 720 accctccaga tatgccctgc gtccaagcccaatatagccc ttcccctcca ggttccagtt 780 atgcggcgca gacatacagc tcggaatacaccacggagat catgaacccc gactacacca 840 agctgaccat ggaccttggc agcactgagatcacggctac agccaccacg tccctgccca 900 gcatcagtac cttcgtggag ggctactcgagcaactacga actcaagcct tcctgcgtgt 960 accaaatgca gcggcccttg atcaaagtggaggaggggcg ggcgcccagc taccatcacc 1020 atcaccacca ccaccaccac caccaccaccatcaccagca gcagcatcag cagccatcca 1080 ttcctccagc ctccagcccg gaggacgaggtgctgcccag cacctccatg tacttcaagc 1140 agtccccacc gtccaccccc accacgccggccttcccccc gcaggcgggg gcgttatggg 1200 acgaggcact gccctcggcg cccggctgcatcgcacccgg cccgctgctg gacccgccga 1260 tgaaggcggt ccccacggtg gccggcgcgcgcttcccgct cttccacttc aagccctcgc 1320 cgccgcatcc ccccgcgccc agcccggccggcggccacca cctcggctac gacccgacgg 1380 ccgctgccgc gctcagcctg ccgctgggagccgcagccgc cgcgggcagc caggccgccg 1440 cgcttgaggg ccacccgtac gggctgccgctggccaagag ggcggccccg ctggccttcc 1500 cgcctctcgg cctcacgccc tcccctaccgcgtccagcct gctgggcgag agtcccagcc 1560 tgccgtcgcc gcccagcagg agctcgtcgtctggcgaggg cacgtgtgcc gtgtgcgggg 1620 acaacgccgc ctgccagcac tacggcgtgcgaacctgcga gggctgcaag ggctttttca 1680 agagaacagt gcagaaaaat gcaaaatatgtttgcctggc aaataaaaac tgcccagtag 1740 acaagagacg tcgaaaccga tgtcagtactgtcgatttca gaagtgtctc agtgttggaa 1800 tggtaaaaga agttgtccgt acagatagtctgaaagggag gagaggtcgt ctgccttcca 1860 aaccaaagag cccattacaa caggaaccttctcagccctc tccaccttct cctccaatct 1920 gcatgatgaa tgctcttgtc cgagctttaacagactcaac acccagagat cttgattatt 1980 ccagatactg tcccactgac caggctgctgcaggcacaga tgctgagcat gtgcaacaat 2040 tctacaacct cctgacagcc tccattgatgtatccagaag ctgggcagaa aggattccgg 2100 gatttactga tctccccaaa gaagatcagacattacttat tgaatcagcc tttttggagc 2160 tgtttgtcct cagactttcc atcaggtcaaacactgctga agataagttt gtgttctgca 2220 atggacttgt cctgcatcga cttcagtgccttcgtggatt tggggagtgg ctcgactcta 2280 ttaaagactt ttccttaaat ttgcagagcctgaaccttga tatccaagcc ttagcctgcc 2340 tgtcagcact gagcatgatc acagaaagacatgggttaaa agaaccaaag agagtcgaag 2400 agctatgcaa caagatcaca agcagtttaaaagaccacca gagtaaggga caggctctgg 2460 aacccaacga gtccaaggtc ctggttgccctggtagaact gaggaagatc tgcaccctgg 2520 gcctccagcg catcttctac ctgaagctggaagacttggt gtctccacct tccatcattg 2580 acaagctctt cctggacacc ctacctttctaatcaggagc agtggagcag tgagctgcct 2640 cctctcctag caccctgctt ctacgcagcaaagggatagg tttggaaacc tatcatttcc 2700 tgtccttcct taagaggaaa agcagctcctgtagaaagca aagactttct tttttttctg 2760 gctcttttcc ttacaaccta aagccagaaaacttgcagag tattgtgttg gggttgtgtt 2820 ttatatttag gcattggggg atggggtgggagggggttat agttcatgag ggttttctaa 2880 gaaattgcta acaaagcact tttggacaatgctatcccag caggaaaaaa aaggataata 2940 taactgtttt aaaactcttt ctggggaatccaattatagt tgctttgtat ttaaaaacaa 3000 gaacagccaa gggttgttcg ccagggtaggatgtgtctta aagattggtc ccttgaaaat 3060 atgcttcctg tatcaaaggt acgtatgtggtgcaaacaag gcagaaactt ccttttaatt 3120 tccttcttcc tttattttaa caaatggtgaaagatggagg attacctaca aatcagacat 3180 ggcaaaacaa taatggctgt ttgcttccataaacaagtgc aattttttaa agtgctgtct 3240 tactaagtct tgtttattaa ctctcctttattctatatgg aaataaaaag gaggcagtca 3300 tgttagcaaa tgacacgtta atatccctagcagaggctgt gttcaccttc cctgtcgatc 3360 ccttctgagg tatggcccat ccaagacttttaggccattc ttgatggaac cagatccctg 3420 ccctgactgt ccagctatcc tgaaagtggatcagattata aactggatta catgtaactg 3480 ttttggttgt gttctatcaa ccccaccagagttccctaaa cttgcttcag ttatagtaac 3540 tgactggtat attcattcag aagcgccataagtcagttga gtatttgatc cctagataag 3600 aacatgcaaa tcagcaggaa ctggtcatacagggtaagca ccagggacaa taaggatttt 3660 tatagatata atttaatttt tggtaattgggttaaggaga ccaattttgg agagcaagca 3720 aatcttcttt ttaaaaaata gtatgaatgtgaatactaga aaagatttaa gaaatagtat 3780 gagtgtgagt actaggaagg at 3802 24400 DNA RAT 2 ccgagtctcc tgcctcccgc cccccacccc tccagcgcct gctcctcctccgctccccat 60 acacagacac gctcacaccc gctccttcac ttgcacacac agacacacgcgcgctcacac 120 gctccgcaca cacactccac tctctcccgc gcgctcacac ccctctctctcggcgccctc 180 gccggtgtcg cgccgcgccg cgccgcagcc ggacgcccct ccagggctcactttgcaacg 240 ctgacagagc gggcagtggc cgtggaggtg ggaaacgtgg cgacatcctagcccctggtc 300 gcagccggag actggacgct gcggaacctc tcggcggcgc tctcccatgagttgggatcg 360 cagcatcccc agccagccgc tgctcaccgc ctctgggagc cgctgggtttgtgcaccgca 420 gcccttccgg gacagcagct gtgactctcc cccaatccag atttcggggtcgctctctag 480 aaactcgctc taaagacgga acctccacag aacccaaagc ccactgcgggagagcgcagc 540 ccgacaagcc cgggcgctga gcctggaccc tcaacagagc gggccagcacagcggcggcg 600 gctgcttcgc ctatcccgac gtccccgcct cctacactct cagcctccgctggagagacc 660 cccagcccca ccattcagcg cgcaagatac cctccagata tgccctgcgtgcaagcccaa 720 tatagccctt cgcctccggg gtccacttat gccacgcaga cttatggctcggaatacacc 780 acagaaatca tgaaccccga ctatgccaag ctgaccatgg acctcggtagcacggggatc 840 atggccacgg ccacgacgtc cctgcccagc ttcagtacct tcatggagggctaccccagc 900 agctgcgaac tcaagccctc ctgcctgtac caaatgccgc cttctgggcctcggcctttg 960 atcaagatgg aagagggtcg cgagcatggc taccaccacc accaccaccatcaccatcat 1020 catcaccacc accaccagca gcagcagccg tccattcctc ctccctctggccccgaggac 1080 gaggtactgc ccagcacctc catgtacttc aagcagtctc cgccgtctacgccgaccact 1140 ccaggcttcc ccccgcaggc gggggcgctg tgggacgacg agctgccctctgcgcctggc 1200 tgcatcgctc cgggaccgct gctggacccg cagatgaagg cagtgcccccaatggccgct 1260 gctgcgcgct tcccgatctt cttcaagccc tcaccgccac accctcccgcgcccagccca 1320 gccggcggcc accacctggg ctatgacccc acggccgcag ctgcgctcagtctacccctg 1380 ggagccgcgg ccgccgcggg cagccaagct gctgcgctcg agggccatccgtacgggctc 1440 ccgctggcca agaggacggc cacgttgacc ttccctccgc tgggcctcacagcgtcccct 1500 accgcgtcca gcctgctggg agagagcccc agcctaccat cgccacccaataggagctca 1560 tcatccggcg agggcacgtg tgctgtgtgc ggggacaatg ctgcctgccagcactacgga 1620 gtccgcacct gcgagggctg caagggcttc ttcaagagaa cggtgcagaaaaacgcaaaa 1680 tatgtttgct tggcaaataa aaactgcccg gtagacaaga gacgtcgaaatcgatgtcag 1740 tactgcaggt ttcagaagtg tctcagtgtc gggatggtga aggaagttgtgcgtacagat 1800 agtctgaaag ggaggagagg tcgtctgcct tccaaaccaa agagcccactacaacaggag 1860 ccctcgcagc cctccccacc atctcctccg atctgtatga tgaacgcccttgtccgagct 1920 ttaacagacg caacgcccag agaccttgat tactccagat actgtcccaccgaccaggcc 1980 actgcgggca cagacgctga gcacgtgcag cagttctaca accttctgacggcctccatc 2040 gacgtgtcca gaagctgggc agaaaagatc cccggattca ctgatctccccaaagaagat 2100 cagacgttac ttatagaatc agcctttttg gagctgttcg ttcttagactttctatcagg 2160 tcaaacactg ctgaagataa gtttgtgttc tgcaatggac ttgtcctgcaccgacttcag 2220 tgccttcgcg gatttgggga gtggctcgac tccattaaag acttttctttaaatttgcag 2280 agcctgaacc ttgatatcca agccttagcc tgcctgtcag cactgagtatgatcacagag 2340 cgacatgggt taaaagaacc aaagagagtg gaggagctat gcaacaagatcacaagcagc 2400 ttaaaggacc accagaggaa gggacaggct ctggagccct cagagcccaaggtccttcgc 2460 gcactggtgg aactgaggaa gatctgcacc cagggcctcc agcgtatcttctacctgaag 2520 ctggaggact tggtgtcccc accttctgtc atcgacaagc tcttccttgataccctgcct 2580 ttctgagcag gggaagcctg agcagagagc tacttgctct gctggcactggtcattaagt 2640 gagcaaaagg atgggtttga acacctgccc ctctatcctt cctccaggggaaaaagcagc 2700 tcccatagaa agcaaagact tttttttttc ctggcacctt tccttacaacctaaagccag 2760 aaaccttgca gagtattgtg ttggggttgt gttttatatt taggctttggtgggtgggct 2820 gggagggggt aaaatagttc atgaggcttt tctaagaaat tgctgacgaagcacttttgg 2880 atgatgctat cccagcagtg gggtggggag aaaggataat ataactgttttaaaaactct 2940 ttccggggga atatgactat ggttgctttg tatttaaaaa taagaacagccaagggctgt 3000 tttaccaggg tagggctgtg tcttaagact gatcccttta gtatgtacttcccggatcga 3060 ggcacataag tggtgcaaat gaggcgggga aattcttcat ttcttcatttctttcttctt 3120 cttaaaataa aatggcaaaa aaaaaaagat ggaagattat ctacaaatcagacttagcaa 3180 aatgataatg gctattcgct tccacataca agtgcaattt tttagagtgctgtcttacta 3240 agtcttgttt gtgaactctc cctcatttta tatgaaaata agaaggaggcagtcatgtta 3300 tcaaacggcg tgctcatttt cctagctcac ccttggtcca cctgccctgtagaacccttc 3360 ggaggtatgg cccttctaag actttcaggc cactcttgat ggaattcgacacccctcccc 3420 tcaacccatg actatccaga tgtcctgaat ggggatcagg ttataaaatggattgcatat 3480 gactgtgttc gctgtgtgtt tgtcaacctg gacagagttc tctaaaccttctttagttgt 3540 agcaagttcc tgattcctcc attcagaagc ccaaggagca ttgggtgactcgatcaaggg 3600 ttaaccctag gagaacatgc aaataagtag gaactgggtc agacagggtaagcaccagag 3660 atgataagga tttatatata aatatatata aaattaattt ttgttattggttatagacaa 3720 ttttggaaag caagagaatc atctcttttt tttttttaaa gaggaaaagatagtattgat 3780 gtattagcaa agattagtgg ggtacggttc aacattccgt gtttgtgcccccttttctat 3840 gtttctactg ttgatggcat attattatga aatgattcgt tgcatagtgtccttatttgt 3900 atgaacattt gtatgcacgt tctattgtaa tcgctttgcc tgtatttattgcaagaccac 3960 cagctcctgg aggctgagtt acagaataat caaatggggt gttcgtggtgacttggatac 4020 accggttaga aattaaataa gcatatatat atatataaaa acatagcaggttacatatat 4080 atttataatg tgtcttttta ttaaccattt gtacaataaa tgtcacttcccacgcagtta 4140 ttttatcctt tgtttgcagt gacctttaag gcagcactgt ttagcactttgatatgaaat 4200 tttttgctta tttttttgct aaattcaaat aacgtttgaa gatttttaggtctaaaagtc 4260 tttatattat atacactgta tcaagtcaag atacctttgg ccgttttgctaagactcaaa 4320 ctttgaatgt caaaccaatg tcacggtagc ttctgttagc ttttaatcatttttgcttta 4380 gtcttttttt ttaaaaaaaa 4400 3 349 DNA Hepatitis A virus 3caatcactct gatgagtatt tgtcatttag ttgttattta tctgtcacag aacaatcaga 60gttttatttt cccagagctc cattgaattc aaatgctatg ttatccactg aatcaatgat 120gagtagaatt gcagctggag atttggagtc atcagtggat gatcctagat cagaggagga 180taaaagattt gagagtcaca tagaatgcag gaagccatat aaagaactga gattagaagt 240agggaaacaa agactcaagt atgctcagga agaattgtca aatgaagtac ttccaccccc 300taggaaaatg aaggggctgt tttcacaagc caaaatttca cttttttat 349 4 433 DNA Homosapiens 4 taacaatctt gttgcatagc tcttcgactc tctttggttc ttttaacccatgtctttctg 60 tgatcatgct cagtgctgac aggcaggcta aggcttggat atcaaggttcaggctctgca 120 aatttaagga aaagtcttta atagagtcga gccactcccc aaatccacgaaggcactgaa 180 gtcgatgcag gacaagtcca ttgcagaaca caaacttatc ttcagcagtgtttgacctga 240 tggaaagtct gaggacaaac agctccaaaa aggctgattc aataagtaatgtctgatctt 300 ctttggggag attagtaaat cccggaatct tttctgccca gcttctggatacatcaatgg 360 aggctgtcag gaggttgtag aattgttgca catgctcagc atctgtgcctgcagcagcct 420 ggtcagtggg aca 433 5 414 DNA Suaeda maritimamodified_base (127)..(413) N = A, C, G and/or T/U 5 ctcatccttcactctttcta attttctctc tttcatttcc tcacatacat tttattccca 60 ttttgtcccttgctctttca ttatcaaaac atacaaactt aattttatta ttataaatta 120 gcctctnanncncctccant tntttttnca nacttgagnn naattccgtt ttatacagcg 180 gntgaagaaaaagaaaaaga aaataatgac aaagcaagaa atggtggttt cagaagaagg 240 aaaagcgaattcaagcaaat catcatcatc aacatcatca tgtacatntn aaaaaccacc 300 accagngccaagcaaataca tatnagnacc aatgttngaa tgngagaggc aactgnttaa 360 agacctggatatnaatggng gntnttgggt tgattntatg agagcttntt ntnc 414 6 1606 DNA Susscrofa 6 ataccctcca gatatgccct gcgtgcaagc ccagtatagc ccttcgcctccaggttccag 60 ttatgcagcc cagacatatg gctcggaata caccacagag atcatgaatcctgactacac 120 caagctgacc atggaccttg gcagcaccga gatcacggcc actgctacaacgtccctgcc 180 cagcttcagt accttcatgg agggctactc cagcaactac gaactcaagccttcctgcct 240 gtaccaaatg cagccgtcgg ggcctcggcc cctgatcaag atggaggagggccgcgcgca 300 cggctaccac catcaccacc acgaccacca ccaccatcac caccaccagcagcagcagca 360 gcagcagcag ccacctccgc agcagcagca gccatccatt ccgcccccctccggtccgga 420 ggacgaggtg ctgcccagca cctctatgta cttcaagcag tccccgccgtccacccccac 480 cacgccggtc ttcccccagc aggcgggggc gctgtgggaa gacgcgctgccctctgcgca 540 gggctgcatc gcgcccggcc cgctgctcga cccgccgatg aaggcggtgcccacggtggc 600 cggcgcgcgc ttccctctct tccacttcaa gccctcgccg ccgcatccgcccgcgcccag 660 ccccgccggc ggccaccatc tcggctacga cccgacggcc gctgccgcgcttggcctgcc 720 gctaggagcc gccgccgccg ccgccgccgc cgccgcaggc agtcaggccgccgcgctcga 780 gggccacccg tacgggctgc cactggccaa aagggcggcc gcgctggccttctcgccgct 840 gggcctcacg acctccccca ccacgtccag cctactgggc gagagccccagcctgccgtc 900 tccgcccaac aggagcaccg cgtcaggcga ggggacgtgc gccgtgtgcggggacaacgc 960 cgcctgtcag cactatggcg tgcgcacctg cgagggctgc aagggcttcttcaagagaac 1020 ggtgcagaaa aatgcaaaat atgtttgcct ggcaaataaa aactgccccgtagacaagag 1080 gcgtcgaaac cgatgtcagt actgtcgatt tcagaagtgt ctcagtgtcggaatggttaa 1140 agaagttgtc cgtacagata gtctgaaagg gaggagaggt cggctgccttccaaaccaaa 1200 gagcccgtta cagcaggaac cctctcagcc ctctccacct tctcctccggtctgtatgat 1260 gaatgccctt gtccgagctt taacagactc aacgcccaga gatcttgattattccagagg 1320 ccacgcctgc ggcatatgga agttcccagg ctaggagtcg aatcagagatgcagctgcca 1380 gcctacgcca cagacacagc cacgtgggat ctgagccgtg tctgcagtccacaccacagc 1440 ttgtggcaaa accagatcct taacctatgg agtgaggcca agggatcgaaccttcatcct 1500 cacagatact agacggcttc ataacccatt gagccacagg aggagctccacccagagatc 1560 ttaataaaac agtttcttgg gcctaaaaaa aaaaaaaaaa aaaaaa 16067 1967 DNA Homo sapiens 7 tggcgccggc cggggtcccg gccaaccgcc gaatttagtaacatcgcctg cgtcaatcac 60 gcgcctcggt gcgtcaggcc gcgcggctcc aggtcctgctcccccccttc aagcctttga 120 atggatacaa tgtagcagcg ccctccttcc ttccgaggctggattggaac cgccgcagtg 180 cagagactcg gttgctctcg gctgggtcaa ctttcggggcattctcccac gatcctctcc 240 gcaccaccgt gtctgaattg gaagtggagg cgaagaaagatatacatgcc atatttacct 300 atatgtagtt tgttttcaag tttctggtcc tagctcgaaccttcttcgat tctgaaatgt 360 gtgctgtcta caaaggaatc ttgtatctcc cctcggcgcagccccccgcc ccgccacaca 420 cacacaaatt gggacaggtc aaacatataa aacggtatttgtgattcaag cggaccacat 480 ggggaccact ctatctgcat tgtttcactc aaatattttctcctgtccaa aaattcattt 540 ctgaaagaga ctgcgttcac tcagcagcaa cctttgggactaggggtctt taactctgat 600 aaattttgtt ttcatcaaga aatttacact taaatttatcatttccagga agaaattgct 660 ctccttcata cagtcaccca ggctttcggc acaccatttcatgacaaatg tgtccgagga 720 gaccaaagca aatcccctag cgagggactg actaataagtcctgttgatt gatttcgaaa 780 tgtttaattt gggagatgtg ggcggagggc atctacaaccatcaaaaagt gaaagtgcta 840 gttgagagtt ccatttctga cccggtgccg gggaggaggaatgatttgca atagtcagac 900 ccgctcagct gttcaacacg tgtgtgtttg ttttacacacagagtagttt ctgctgcagc 960 cgcgtgtgca tgatggatgt gcacttcgct gggttataacgtgtccagtt aagaaaccca 1020 cgccgtacgt gtaaagaaat caaaccttat ccccggaaccatctgcatcc ctgtgtgaac 1080 acgcacccag taaatgatgc ggggaggggg gattagcctgggcgcagagg accggagcaa 1140 cgtaaacagc tttagaacct atgcaagagg aaagtgcagctgcacctcag ggcgtcttcg 1200 ggctggtgcc agacgccttc tgcaccggct gccaggtcactggagctggt cagaagctgg 1260 ctggcggagc ttccctttcg gaagagctgt cctctcccttacccccctcg ccctggctcc 1320 gtgcctcggg gcagcctcgg aggcgcgcca gcagcactcctccaactcta ctccacccga 1380 gcctgacagc tgggcggtcc cgcctgaccc gtgggcaggccgctgcaccc tcccgcagac 1440 gcacgccctg gcgagcggtt ccgctgcaaa aagagaagcccccaggccgg ggccggccgt 1500 gcggcggagt ttccattgtg cggccgtgcg actggccgaggaacgcgcgc gcgcgcgcac 1560 acgaacacac acaccctccc tcgcacacgc ggaaccggctgggccagggg agggaggagg 1620 agggtgacgt agcgtcccat ggcgtcacat tgacgtctcgcattccaggc actctatgga 1680 gaggccgcta gggctcctgt ggcataaatg acgtgccgagagagcgagcg aacgcgcagc 1740 cgggagagcg gagtctcctg cctcccgccc cccacccctccagctcctgc tcctcctccg 1800 ctccccatac acagacgcgc tcacacccgc tccctcactcgaacacacag acacaagcgc 1860 gcacacaggc tccgcacaca cacacttcgc tctcccgcgcgctcacaccc ctcttgccct 1920 gagcccttgc cggtgcagcg cggcgccgca gctggacgcccctcccg 1967 8 5115 DNA Sus scrofa 8 ggcagcgccg ccgccgcttc gcctcgcaggacgtccccgc ctcctccact ctcagcctct 60 gctggagaga cccccagccc caccattcagcgcgcaagat accctccaga tatgccctgc 120 gtgcaagccc agtatagccc ttcgcctccaggttccagtt atgcagccca gacatatggc 180 tcggaataca ccacagagat catgaatcctgactacacca agctgaccat ggaccttggc 240 agcaccgaga tcacggccac tgctacaacgtccctgccca gcttcagtac cttcatggag 300 ggctactcca gcaactacga actcaagccttcctgcctgt accaaatgca gccgtcgggg 360 cctcggcccc tgatcaagat ggaggagggccgcgcgcacg gctaccacca tcaccaccac 420 gaccaccacc accatcacca ccaccagcagcagcagcagc agcagcagcc acctccgcag 480 cagcagcagc catccattcc gcccccctccggtccggagg acgaggtgct gcccagcacc 540 tctatgtact tcaagcagtc cccgccgtccacccccacca cgccggtctt cccccagcag 600 gcgggggcgc tgtgggaaga cgcgctgccctctgcgcagg gctgcatcgc gcccggcccg 660 ctgctcgacc cgccgatgaa ggcggtgcccacggtggccg gcgcgcgctt ccctctcttc 720 cacttcaagc cctcgccgcc gcatccgcccgcgcccagcc ccgccggcgg ccaccatctc 780 ggatacgacc cgacggccgc tgccgcgcttggcctgccgc taggagccgc cgcsgccgcc 840 gccgccgccg ccgcaggcag tcaggccgccgcgctcgagg gccacccgta cgggctgcca 900 ctggccaaaa gggcggccgc gctggccttctcgccgctgg gcctcacgac ctcccccacc 960 acgtccagcc tactgggcga gagccccagcctgccgtctc cgcccaacag gagcaccgcg 1020 tcaggcgagg ggacgtgcgc cgtgtgcggggacaacgccg cctgtcagca ctatggcgtg 1080 cgcacctgcg agggctgcaa gggcttcttcaagagaacgg tgcagaaaaa tgcaaaatat 1140 gtttgcctgg caaataaaaa ctgccccgtagacaagaggc gtcgaaaccg atgtcagtac 1200 tgtcgatttc agaagtgtct cagtgtcggaatggttaaag aagttgtccg tacagatagt 1260 ctgaaaggga ggagaggtcg gctgccttccaaaccaaaga gcccgttaca gcaggaaccc 1320 tctcagccct ctccaccttc tcctccggtctgtatgatga atgcccttgt ccgagcttta 1380 acagactcaa cgcccagaga tcttgattattccagatact gccccgctga ccaggccgct 1440 gcaggcacag atgctgagca tgtacaacagttctacaacc ttctcacagc ctccattgac 1500 gtatccagaa gctgggcaga aaagattcctggatttactg atctccctaa agaagatcag 1560 acattactta tagaatcagc ctttttggagctgtttgttc tcagactttc catcaggtca 1620 aacactgctg aagataagtt tgtgttctgcaatggacttg tcctgcatcg acttcagtgc 1680 cttcgtggat ttggggagtg gctcgactccattaaagact tttccttacg tttgcagagc 1740 ctgaaccttg atatccaagc cttagcctgcctgtcagcac tgagcatgat cacagaacga 1800 catgggttaa aagaaccaaa gagagtggaggagctatgca acaagatcac aagcagctta 1860 aaagagcacc agagtaaggg acaggctttggagcccaccg agcccaaggt cctgcgtgcc 1920 ctggtggaac tgcggaagat ctgcaccctgggcctccagc gcatcttcta cctaaagctg 1980 gaagacttgg tgtctccacc ttccatcatcgacaagctct tcctagatac cctgcctttc 2040 tgagcaggag cagcctgagc agggagctgcttcatctgct agcagccact tgctaagcgg 2100 caaaggaatg ggtctggaca tctaccattttctgtccttc cttaagagaa aaagcagctc 2160 ctgtagaaat aaaagacttc ttcttcttcttctttttttt tttttttttt ttctggcact 2220 tttccttatg actgaaagcc agaaaacttacagagtattg tgttggggtt gtgttttata 2280 tttaggcttt ggggttgggg tgggaggggggtatagttca tgagggtttt ctaagaaatt 2340 gctaacaaag cacttttgga cgatgctatcccagcaggaa aaaaaaaaaa aaaaaggata 2400 atgtaactgt tttaaaactc tttctggggaatacagttat agttgatttg tatttaaaaa 2460 caagaacagc caagggttgt ttgccagggtagggtgtgtt tgagattgat ccctttagaa 2520 tatacttctt gtatcaaggg tacatatgtggtgcaaaaaa agcagaaatt ccctcttcct 2580 aatttccttc ttcatttatt ttaacaaatggtaaaagaag gaggattacc tataaatcgg 2640 acatagcaaa atgataatgg cttttcgcttccatatacaa gtgcaatttt taaagtgctg 2700 tcttactaag tcttgtttat taactctcctttattttata tggaaataaa ggaggcagtc 2760 atgatagcaa atgacacatg ctaattttcctagcagaggc gttgtccacc tgacccatag 2820 aactcttctg agacgtggtc catccaagatttttggccgt tcttgatgga tccgggtccc 2880 tgccctgagc tgctttgaaa ggggctcagattatatgagg ggttacgtac agcttttttt 2940 gtcgctttct ataaatccag ccagatttccctaaacttgc ttcaattatg gtaacagact 3000 gacacattca ttcagaagcc ccaagagcattcagtgaatt tcaagtgttt gaccccaaga 3060 taagaacatg caaataagta agaatggatcatacagggta agcaccaggg ataataagtt 3120 ttttaaatat atataattta atttttattatcagttaaag agacaatttt ggagagcaag 3180 tgattcttat taaaaaatta gtgtgaatgtgagtactaga aaggattagt gggctgcgtt 3240 tcaacattcc gtgttcgtac tcccttttgtatgtttatac tgttaatgcc atattattat 3300 gagataattt gttgcatagt gtcctttatttgtataaaca tttgtatgca cgttatattg 3360 taatagcttt gcctgtattt attgcaagaccaccagctcc tggaagctga gttacagagt 3420 acttaaatgg ggtgttcaca gtgaatttggatacaccaat tagaaattaa ataagcaaat 3480 atatatataa atatagcaag ttacatatatatttataatg tgtcttttta ttaaccattt 3540 gtacaataaa tgttacttcc catgcagttattttacggtt catttgcagt gacttttaag 3600 gcagtactgt ttagcacttt gatattaaaattttgcttac gttttgctaa attcaaatga 3660 tgtttgaaga tttttaggtc taaaagtctttatattatat actctgtatc aagtcaaaat 3720 atctttggtc attttgctaa gaaacaaactttgaatgtca aactgatgtc atagtagttt 3780 ttgttagctc tgaatcattt ttgctttagtctttttaaag gaaaaataac aaaactatgc 3840 tgtttatatt gtcattaaat tatacaatcaaacaaatgcc aaatgaattg cctaattgct 3900 gcaatgaata acccagatag caaaatcatgtatgtttttt tcccaaaatt cattctgata 3960 tttgatcatt atgtttgtgt gagcttttatgaaagattat tatttttata tcaagatgat 4020 aggatctgga atgttaggat ctcagaatgttagatttaaa aggggcctgc cttgtcaact 4080 agtccacccc cacacactaa aattcatagaggaggaaatt ggggcccagc aaagggcaaa 4140 gggttaacca aggacaaaga gctggtaacagaatcaagac caggacctaa ttctccttgc 4200 cacagtcttt ttactcactt tactgcatctgtaggaaaac aggcttttaa aaataaccag 4260 taatatgtac attttaccgt gagtaaagcagtaactttgc agtaattttt gagcttatat 4320 gcaaacataa tgaacattat taaatatcaggagagctaac atttcatacg agttagcttc 4380 agaccaaatt caaattgaat ttgaataaattagaaatact gtgcatacat aacccttttg 4440 tgcactgtta agttttgaaa tcttaatcggtttttgtttt ttgtttttgt catgtctgta 4500 aaggaaaaac aaaagaaaaa aacagagccctggagaaatg ctgtcacttt ttatttttac 4560 acccatcaga tttaaggaaa agactttttagctgatattg attggttgga aggaatgaag 4620 aaggttttta cttataggtc caggcactagtgctgaaaat aaagattata gccagtgttc 4680 ttctgtcttc catagttatt acaactatgagagccccccc aagtcatcta tcaattcaac 4740 tctctttttt ctttttgtct taatgttgacacacaagttt atacagagtg gatgaccaga 4800 ctagctcaga agaggacagc aagaattaaagcaggtgatt cttcccttgt gggagagctc 4860 tctcagtgtg aacatgcctt ctttgggcagaaatcaggaa tccaccagct gttaatggag 4920 agtgccttgc ttttatttca gacagcagagttttccaaag tttctctgct cctctaacag 4980 cgttgctctt tagtgtgtgt taacctgtgtttgaaagaaa tgctcttgta cattaacaat 5040 gtaaatttaa atgattaaat taaattacattttatcaatg gctaaaaaaa aaaaaaaaaa 5100 aaaaaaaaaa aaaaa 5115 9 1453 DNAHomo sapiens 9 gtaggtccga aggcaagacc cttttctcct ccctggctga gggaagtgggtgggggaacc 60 acacactcgg cgggcagcgt ggtcgacctg cccagtgcca ggacagtgactgctggccgc 120 gaatttcaca acacaggtgg cttcctcaca ggaagctcct ctgtataccacaccctgttg 180 ctactgagtg gagcagccaa attaaattaa gcttgcattg ctcaaaattaattttcctaa 240 gagaaataca aatacaccaa tagattaggg tattttatac atttttaatttcatttttgc 300 tcttctttta tagccagtgt gcacatgtaa gagttatgga atcacttagagcaatactga 360 gcattttcat ttatataaaa cccaaatcat ttgggtgcag aagtttggatgattgaagct 420 cagagggaag gagaaaagca tttgagatga aaccaaagag taaatttgagtttggcaaag 480 aacagattgc acttttggtt tgtaccacct ctttacaaat ttgttaaaggatactaggtg 540 ccaagcctgt gtgggcacca tagataatgc agataaatag aatacaatcccagcaagctc 600 atcttctagc tagaaacctc agaaagacac aaataagagc agtaaaaaggggataacgca 660 ggaaagaaca tgtgagcaaa aggcctggag aaatcctaga ggtgctgaagggagaaggag 720 aatagatcaa agatcaggca gtgttctttc ttttaaatga ttagcctttcatttcatccc 780 aacaactgga cagcaagata agtaactgga cttcaaacta gtgagtgtatttttaaggcc 840 ctgcttgtta aagaaaggct tgaactggcc tctcctcatc actgcttcttccaacaggcc 900 ctcatcacct tttttcaagt caagatttca tcccatacat gcatgactcaatcagatttg 960 gaaatgtggg taagagaaag atgtcaaagg aaatgtgaag tattcacttctctattagtc 1020 acacctttta caccatagac tccaaagagg cgttaagcac ctggttttcctttggctcag 1080 aaaaaccaac caccaaaaac cgccgttttt taccatttat atttagccataaagaaagaa 1140 aaataattag ataaatcatc cactacatcc aataattctc agcgccttctcactcagttc 1200 agcctctctg aacaatagta agcaccctgg ataccagcca ctttgggggcaacatagtca 1260 aactggcaga gaaatcaagt ctattgagaa actgcttttt ttcatgggtactaattctag 1320 tgtcataagg aaatacctat actaacttgc ctattatgat agttataaactgttatcaca 1380 aaacagtcac tgatatgttt tattagttag aattgggata tatatatgtgtgtgtgtgtg 1440 tgtgtgtgtg tgt 1453 10 1703 DNA Homo sapiens 10tggcgccggc cggggtcccg gccaaccgcc gaatttagta acatcgcctg cgtcaatcac 60gcgcctcggt gcgtcaggcc gcgcggctcc aggtcctgct cccccccttc aagcctttga 120atggatacaa tgtagcagcg ccctccttcc ttccgaggct ggattggaac cgccgcagtg 180cagagactcg gttgctctcg gctgggtcaa ctttcggggc attctcccac gatcctctcc 240gcaccaccgt gtctgaattg gaagtggagg cgaagaaaga tatacatgcc atatttacct 300atatgtagtt tgttttcaag tttctggtcc tagctcgaac cttcttcgat tctgaaatgt 360gtgctgtcta caaaggaatc ttgtatctcc cctcggcgca gccccccgcc ccgccacaca 420cacacaaatt gggacaggtc aaacatataa aacggtattt gtgattcaag cggaccacat 480ggggaccact ctatctgcat tgtttcactc aaatattttc tcctgtccaa aaattcattt 540ctgaaagaga ctgcgttcac tcagcagcaa cctttgggac taggggtctt taactctgat 600aaattttgtt ttcatcaaga aatttacact taaatttatc atttccagga agaaattgct 660ctccttcata cagtcaccca ggctttcggc acaccatttc atgacaaatg tgtccgagga 720gaccaaagca aatcccctag cgagggactg actaataagt cctgttgatt gatttcgaaa 780tgtttaattt gggagatgtg ggcggagggc atctacaacc atcaaaaagt gaaagtgcta 840gttgagagtt ccatttctga cccggtgccg gggaggagga atgatttgca atagtcagac 900ccgctcagct gttcaacacg tgtgtgtttg ttttacacac agagtagttt ctgctgcagc 960cgcgtgtgca tgatggatgt gcacttcgct gggttataac gtgtccagtt aagaaaccca 1020cgccgtacgt gtaaagaaat caaaccttat ccccggaacc atctgcatcc ctgtgtgaac 1080acgcacccag taaatgatgc ggggaggggg gattagcctg ggcgcagagg accggagcaa 1140cgtaaacagc tttagaacct atgcaagagg aaagtgcagc tgcacctcag ggcgtcttcg 1200ggctggtgcc agacgccttc tgcaccggct gccaggtcac tggagctggt cagaagctgg 1260ctggcggagc ttccctttcg gaagagctgt cctctccctt acccccctcg ccctggctcc 1320gtgcctcggg gcagcctcgg aggcgcgcca gcagcactcc tccaactcta ctccacccga 1380gcctgacagc tgggcggtcc cgcctgaccc gtgggcaggc cgctgcaccc tcccgcagac 1440gcacgccctg gcgagcggtt ccgctgcaaa aagagaagcc cccaggccgg ggccggccgt 1500gcggcggagt ttccattgtg cggccgtgcg actggccgag gaacgcgcgc gcgcgcgcac 1560acgaacacac acaccctccc tcgcacacgc ggaaccggct gggccagggg agggaggagg 1620agggtgacgt agcgtcccat ggcgtcacat tgacgtctcg cattccaggc actctatgga 1680gaggccgcta gggctcctgt ggc 1703 11 785 DNA Homo sapiens 11 tgccagcactacggcgtgcg aacctgcgag ggctgcaagg gctttttcaa gagaacagtg 60 cagaaaaatgcaaaatatgt ttgcctggca aataaaaact gcccagtaga caagagacgt 120 cgaaaccgatgtcagtactg tcgatttcag aagtgtctca gtgttggaat ggtaaaagaa 180 gttgtccgtacagatagtct gaaagggagg agaggtcgtc tgccttccaa accaaagagc 240 ccattacaacaggaaccttc tcagccctct ccaccttctc ctccaatctg catgatgaat 300 gctcttgtccgagctttaac agactcaaca cccagagatc ttgattattc cagagtaagt 360 tttatgatttcctgctttca aatgaatgat cagggtctct atttatggct actagtaata 420 agagttgattgaatgatttt gtgtctggca ccatgttaga cagttttcat actttttcta 480 tatttctcgcttcatttagc aattcagtgc atccattgca gcaaataatt tttgccttat 540 tgaatctctaaatgccttaa caagtgaccc tgacagtgct gcacctgtca tacacattgt 600 tgcaggattcctggtggttg tgccaatgaa aatctgcaca gacaaactac aatttgtaga 660 tttatctcgtgatctagaca aagtgactac tgtttttttt catattgtgt tcaaaccatc 720 tgggtgagcctcaagttatt actaagcagt ttatccaatt gcatcagcat tgattgacct 780 gctgc 785 12966 DNA Homo sapiens 12 gtgtgtatat atatatatat gggtgggtgt tttgttgcagctgctgatct ttttctttgc 60 agatggtaca aactctcccg agtcaatttc ctgggcctatgtccccacct agctgactga 120 agttatcaac aggggtccag tttgtgcagg ctgctagccctattggaaga gtggggacga 180 ggtgggagaa agcaaccaca acgtgtgtgg gcaacctcaattggcactca taaaatgtta 240 gaatgtcaac tctctccctt ggccactaaa tctctcacagggtagttttt cttgcctaac 300 tcaggtttac aaatcaatgt gtatgccttg ggggaccaatggcctctttc ctcccaaata 360 aaccactggc tttctctttg tccccctagg ttatagctgaggagcccact ccaattagtt 420 tataggattc aaagcctctt tttaaaaaca tctctgagcttatgaggaaa gacttcaagt 480 ttcccaaatc tagtggagga cagggcaagg gaggaaagataggtacagga gtccacagga 540 ggccaggttt tggcacccct ttgtcaggaa ttcagcttccttactaggga tgaagaaaat 600 aagtgtgggg ctttgtgtct atgctaccag aaggaggagaggatgacact tcctctctgt 660 ttcccagatt agagaacagt gaacccaatg ctgcctgttggctagaaaac aagtgttaac 720 ttgcttctga aagacccttc tctctgtccc tgcagatatgccctgcgtcc aagcccaata 780 tagcccttcc cctccaggtt ccagttatgc ggcgcagacatacagctcgg aatacaccac 840 ggagatcatg aaccccgact acaccaagct gaccatggaccttggcagca ctgagatcac 900 ggctacagcc accacgtccc tgcccagcat cagtaccttcgtggagggct actcgagcaa 960 ctacga 966 13 625 PRT Homo sapiens 13 Met ProCys Val Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly Ser Ser 1 5 10 15 TyrAla Ala Gln Thr Tyr Ser Ser Glu Tyr Thr Thr Glu Ile Met Asn 20 25 30 ProAsp Tyr Thr Lys Leu Thr Met Asp Leu Gly Ser Thr Glu Ile Thr 35 40 45 AlaThr Ala Thr Thr Ser Leu Pro Ser Ile Ser Thr Phe Val Glu Gly 50 55 60 TyrSer Ser Asn Tyr Glu Leu Lys Pro Ser Cys Val Tyr Gln Met Gln 65 70 75 80Arg Pro Leu Ile Lys Val Glu Glu Gly Arg Ala Pro Ser Tyr His His 85 90 95His His His His His His His His His His His His Gln Gln Gln His 100 105110 Gln Gln Pro Ser Ile Pro Pro Ala Ser Ser Pro Glu Asp Glu Val Leu 115120 125 Pro Ser Thr Ser Met Tyr Phe Lys Gln Ser Pro Pro Ser Thr Pro Thr130 135 140 Thr Pro Ala Phe Pro Pro Gln Ala Gly Ala Leu Trp Asp Glu AlaLeu 145 150 155 160 Pro Ser Ala Pro Gly Cys Ile Ala Pro Gly Pro Leu LeuAsp Pro Pro 165 170 175 Met Lys Ala Val Pro Thr Val Ala Gly Ala Arg PhePro Leu Phe His 180 185 190 Phe Lys Pro Ser Pro Pro His Pro Pro Ala ProSer Pro Ala Gly Gly 195 200 205 His His Leu Gly Tyr Asp Pro Thr Ala AlaAla Ala Leu Ser Leu Pro 210 215 220 Leu Gly Ala Ala Ala Ala Ala Gly SerGln Ala Ala Ala Leu Glu Gly 225 230 235 240 His Pro Tyr Gly Leu Pro LeuAla Lys Arg Ala Ala Pro Leu Ala Phe 245 250 255 Pro Pro Leu Gly Leu ThrPro Ser Pro Thr Ala Ser Ser Leu Leu Gly 260 265 270 Glu Ser Pro Ser LeuPro Ser Pro Pro Ser Arg Ser Ser Ser Ser Gly 275 280 285 Glu Gly Thr CysAla Val Cys Gly Asp Asn Ala Ala Cys Gln His Tyr 290 295 300 Gly Val ArgThr Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr Val 305 310 315 320 GlnLys Asn Ala Lys Tyr Val Cys Leu Ala Asn Lys Asn Cys Pro Val 325 330 335Asp Lys Arg Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys 340 345350 Leu Ser Val Gly Met Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys 355360 365 Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Leu Gln Gln370 375 380 Glu Pro Ser Gln Pro Ser Pro Pro Ser Pro Pro Ile Cys Met MetAsn 385 390 395 400 Ala Leu Val Arg Ala Leu Thr Asp Ser Thr Pro Arg AspLeu Asp Tyr 405 410 415 Ser Arg Tyr Cys Pro Thr Asp Gln Ala Ala Ala GlyThr Asp Ala Glu 420 425 430 His Val Gln Gln Phe Tyr Asn Leu Leu Thr AlaSer Ile Asp Val Ser 435 440 445 Arg Ser Trp Ala Glu Arg Ile Pro Gly PheThr Asp Leu Pro Lys Glu 450 455 460 Asp Gln Thr Leu Leu Ile Glu Ser AlaPhe Leu Glu Leu Val Leu Arg 465 470 475 480 Leu Ser Ile Arg Ser Asn ThrAla Glu Asp Lys Phe Val Phe Cys Asn 485 490 495 Gly Leu Val Leu His ArgLeu Gln Cys Leu Arg Gly Phe Gly Glu Trp 500 505 510 Leu Asp Ser Ile LysAsp Phe Ser Leu Asn Leu Gln Ser Leu Asn Leu 515 520 525 Asp Ile Gln AlaLeu Ala Cys Leu Ser Ala Leu Ser Met Ile Thr Glu 530 535 540 Arg His GlyLeu Lys Glu Pro Lys Arg Val Glu Glu Leu Cys Asn Lys 545 550 555 560 IleThr Ser Ser Leu Lys Asp His Gln Ser Lys Gly Gln Ala Leu Glu 565 570 575Pro Asn Glu Ser Lys Val Leu Val Ala Leu Val Glu Leu Arg Lys Ile 580 585590 Cys Thr Leu Gly Leu Gln Arg Ile Phe Tyr Leu Lys Leu Glu Asp Leu 595600 605 Val Ser Pro Pro Ser Ile Ile Asp Lys Leu Phe Leu Asp Thr Leu Pro610 615 620 Phe 625 14 626 PRT Homo sapiens 14 Met Pro Cys Val Gln AlaGln Tyr Ser Pro Ser Pro Pro Gly Ser Ser 1 5 10 15 Tyr Ala Ala Gln ThrTyr Ser Ser Glu Tyr Thr Thr Glu Ile Met Asn 20 25 30 Pro Asp Tyr Thr LysLeu Thr Met Asp Leu Gly Ser Thr Glu Ile Thr 35 40 45 Ala Thr Ala Thr ThrSer Leu Pro Ser Ile Ser Thr Phe Val Glu Gly 50 55 60 Tyr Ser Ser Asn TyrGlu Leu Lys Pro Ser Cys Val Tyr Gln Met Gln 65 70 75 80 Arg Pro Leu IleLys Val Glu Glu Gly Arg Ala Pro Ser Tyr His His 85 90 95 His His His HisHis His His His His His His His Gln Gln Gln His 100 105 110 Gln Gln ProSer Ile Pro Pro Ala Ser Ser Pro Glu Asp Glu Val Leu 115 120 125 Pro SerThr Ser Met Tyr Phe Lys Gln Ser Pro Pro Ser Thr Pro Thr 130 135 140 ThrPro Ala Phe Pro Pro Gln Ala Gly Ala Leu Trp Asp Glu Ala Leu 145 150 155160 Pro Ser Ala Pro Gly Cys Ile Ala Pro Gly Pro Leu Leu Asp Pro Pro 165170 175 Met Lys Ala Val Pro Thr Val Ala Gly Ala Arg Phe Pro Leu Phe His180 185 190 Phe Lys Pro Ser Pro Pro His Pro Pro Ala Pro Ser Pro Ala GlyGly 195 200 205 His His Leu Gly Tyr Asp Pro Thr Ala Ala Ala Ala Leu SerLeu Pro 210 215 220 Leu Gly Ala Ala Ala Ala Ala Gly Ser Gln Ala Ala AlaLeu Glu Gly 225 230 235 240 His Pro Tyr Gly Leu Pro Leu Ala Lys Arg AlaAla Pro Leu Ala Phe 245 250 255 Pro Pro Leu Gly Leu Thr Pro Ser Pro ThrAla Ser Ser Leu Leu Gly 260 265 270 Glu Ser Pro Ser Leu Pro Ser Pro ProSer Arg Ser Ser Ser Ser Gly 275 280 285 Glu Gly Thr Cys Ala Val Cys GlyAsp Asn Ala Ala Cys Gln His Tyr 290 295 300 Gly Val Arg Thr Cys Glu GlyCys Lys Gly Phe Phe Lys Arg Thr Val 305 310 315 320 Gln Lys Asn Ala LysTyr Val Cys Leu Ala Asn Lys Asn Cys Pro Val 325 330 335 Asp Lys Arg ArgArg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys 340 345 350 Leu Ser ValGly Met Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys 355 360 365 Gly ArgArg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Leu Gln Gln 370 375 380 GluPro Ser Gln Pro Ser Pro Pro Ser Pro Pro Ile Cys Met Met Asn 385 390 395400 Ala Leu Val Arg Ala Leu Thr Asp Ser Thr Pro Arg Asp Leu Asp Tyr 405410 415 Ser Arg Tyr Cys Pro Thr Asp Gln Ala Ala Ala Gly Thr Asp Ala Glu420 425 430 His Val Gln Gln Phe Tyr Asn Leu Leu Thr Ala Ser Ile Asp ValSer 435 440 445 Arg Ser Trp Ala Glu Lys Ile Pro Gly Phe Thr Asp Leu ProLys Glu 450 455 460 Asp Gln Thr Leu Leu Ile Glu Ser Ala Phe Leu Glu LeuPhe Val Leu 465 470 475 480 Arg Leu Ser Ile Arg Ser Asn Thr Ala Glu AspLys Phe Val Phe Cys 485 490 495 Asn Gly Leu Val Leu His Arg Leu Gln CysLeu Arg Gly Phe Gly Glu 500 505 510 Trp Leu Asp Ser Ile Lys Asp Phe SerLeu Asn Leu Gln Ser Leu Asn 515 520 525 Leu Asp Ile Gln Ala Leu Ala CysLeu Ser Ala Leu Ser Met Ile Thr 530 535 540 Glu Arg His Gly Leu Lys GluPro Lys Arg Val Glu Glu Leu Cys Asn 545 550 555 560 Lys Ile Thr Ser SerLeu Lys Asp His Gln Ser Lys Gly Gln Ala Leu 565 570 575 Glu Pro Thr GluSer Lys Val Leu Gly Ala Leu Val Glu Leu Arg Lys 580 585 590 Ile Cys ThrLeu Gly Leu Gln Arg Ile Phe Tyr Leu Lys Leu Glu Asp 595 600 605 Leu ValSer Pro Pro Ser Ile Ile Asp Lys Leu Phe Leu Asp Thr Leu 610 615 620 ProPhe 625 15 628 PRT RAT 15 Met Pro Cys Val Gln Ala Gln Tyr Ser Pro SerPro Pro Gly Ser Thr 1 5 10 15 Tyr Ala Thr Gln Thr Tyr Gly Ser Glu TyrThr Thr Glu Ile Met Asn 20 25 30 Pro Asp Tyr Ala Lys Leu Thr Met Asp LeuGly Ser Thr Gly Ile Met 35 40 45 Ala Thr Ala Thr Thr Ser Leu Pro Ser PheSer Thr Phe Met Glu Gly 50 55 60 Tyr Pro Ser Ser Cys Glu Leu Lys Pro SerCys Leu Tyr Gln Met Pro 65 70 75 80 Pro Ser Gly Pro Arg Pro Leu Ile LysMet Glu Glu Gly Arg Glu His 85 90 95 Gly Tyr His His His His His His HisHis His His His His His His 100 105 110 Gln Gln Gln Gln Pro Ser Ile ProPro Pro Ser Gly Pro Glu Asp Glu 115 120 125 Val Leu Pro Ser Thr Ser MetTyr Phe Lys Gln Ser Pro Pro Ser Thr 130 135 140 Pro Thr Thr Pro Gly PhePro Pro Gln Ala Gly Ala Leu Trp Asp Asp 145 150 155 160 Glu Leu Pro SerAla Pro Gly Cys Ile Ala Pro Gly Pro Leu Leu Asp 165 170 175 Pro Gln MetLys Ala Val Pro Pro Met Ala Ala Ala Ala Arg Phe Pro 180 185 190 Ile PhePhe Lys Pro Ser Pro Pro His Pro Pro Ala Pro Ser Pro Ala 195 200 205 GlyGly His His Leu Gly Tyr Asp Pro Thr Ala Ala Ala Ala Leu Ser 210 215 220Leu Pro Leu Gly Ala Ala Ala Ala Ala Gly Ser Gln Ala Ala Ala Leu 225 230235 240 Glu Gly His Pro Tyr Gly Leu Pro Leu Ala Lys Arg Thr Ala Thr Leu245 250 255 Thr Phe Pro Pro Leu Gly Leu Thr Ala Ser Pro Thr Ala Ser SerLeu 260 265 270 Leu Gly Glu Ser Pro Ser Leu Pro Ser Pro Pro Asn Arg SerSer Ser 275 280 285 Ser Gly Glu Gly Thr Cys Ala Val Cys Gly Asp Asn AlaAla Cys Gln 290 295 300 His Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys GlyPhe Phe Lys Arg 305 310 315 320 Thr Val Gln Lys Asn Ala Lys Tyr Val CysLeu Ala Asn Lys Asn Cys 325 330 335 Pro Val Asp Lys Arg Arg Arg Asn ArgCys Gln Tyr Cys Arg Phe Gln 340 345 350 Lys Cys Leu Ser Val Gly Met ValLys Glu Val Val Arg Thr Asp Ser 355 360 365 Leu Lys Gly Arg Arg Gly ArgLeu Pro Ser Lys Pro Lys Ser Pro Leu 370 375 380 Gln Gln Glu Pro Ser GlnPro Ser Pro Pro Ser Pro Pro Ile Cys Met 385 390 395 400 Met Asn Ala LeuVal Arg Ala Leu Thr Asp Ala Thr Pro Arg Asp Leu 405 410 415 Asp Tyr SerArg Tyr Cys Pro Thr Asp Gln Ala Thr Ala Gly Thr Asp 420 425 430 Ala GluHis Val Gln Gln Phe Tyr Asn Leu Leu Thr Ala Ser Ile Asp 435 440 445 ValSer Arg Ser Trp Ala Glu Lys Ile Pro Gly Phe Thr Asp Leu Pro 450 455 460Lys Glu Asp Gln Thr Leu Leu Ile Glu Ser Ala Phe Leu Glu Leu Phe 465 470475 480 Val Leu Arg Leu Ser Ile Arg Ser Asn Thr Ala Glu Asp Lys Phe Val485 490 495 Phe Cys Asn Gly Leu Val Leu His Arg Leu Gln Cys Leu Arg GlyPhe 500 505 510 Gly Glu Trp Leu Asp Ser Ile Lys Asp Phe Ser Leu Asn LeuGln Ser 515 520 525 Leu Asn Leu Asp Ile Gln Ala Leu Ala Cys Leu Ser AlaLeu Ser Met 530 535 540 Ile Thr Glu Arg His Gly Leu Lys Glu Pro Lys ArgVal Glu Glu Leu 545 550 555 560 Cys Asn Lys Ile Thr Ser Ser Leu Lys AspHis Gln Arg Lys Gly Gln 565 570 575 Ala Leu Glu Pro Ser Glu Pro Lys ValLeu Arg Ala Leu Val Glu Leu 580 585 590 Arg Lys Ile Cys Thr Gln Gly LeuGln Arg Ile Phe Tyr Leu Lys Leu 595 600 605 Glu Asp Leu Val Ser Pro ProSer Val Ile Asp Lys Leu Phe Leu Asp 610 615 620 Thr Leu Pro Phe 625 16446 PRT Sus scrofa 16 Met Pro Cys Val Gln Ala Gln Tyr Ser Pro Ser ProPro Gly Ser Ser 1 5 10 15 Tyr Ala Ala Gln Thr Tyr Gly Ser Glu Tyr ThrThr Glu Ile Met Asn 20 25 30 Pro Asp Tyr Thr Lys Leu Thr Met Asp Leu GlySer Thr Glu Ile Thr 35 40 45 Ala Thr Ala Thr Thr Ser Leu Pro Ser Phe SerThr Phe Met Glu Gly 50 55 60 Tyr Ser Ser Asn Tyr Glu Leu Lys Pro Ser CysLeu Tyr Gln Met Gln 65 70 75 80 Pro Ser Gly Pro Arg Pro Leu Ile Lys MetGlu Glu Gly Arg Ala His 85 90 95 Gly Tyr His His His His His Asp His HisHis His His His His Gln 100 105 110 Gln Gln Gln Gln Gln Gln Gln Pro ProPro Gln Gln Gln Gln Pro Ser 115 120 125 Ile Pro Pro Pro Ser Gly Pro GluAsp Glu Val Leu Pro Ser Thr Ser 130 135 140 Met Tyr Phe Lys Gln Ser ProPro Ser Thr Pro Thr Thr Pro Val Phe 145 150 155 160 Pro Gln Gln Ala GlyAla Leu Trp Glu Asp Ala Leu Pro Ser Ala Gln 165 170 175 Gly Cys Ile AlaPro Gly Pro Leu Leu Asp Pro Pro Met Lys Ala Val 180 185 190 Pro Thr ValAla Gly Ala Arg Phe Pro Leu Phe His Phe Lys Pro Ser 195 200 205 Pro ProHis Pro Pro Ala Pro Ser Pro Ala Gly Gly His His Leu Gly 210 215 220 TyrAsp Pro Thr Ala Ala Ala Ala Leu Gly Leu Pro Leu Gly Ala Ala 225 230 235240 Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Gln Ala Ala Ala Leu Glu 245250 255 Gly His Pro Tyr Gly Leu Pro Leu Ala Lys Arg Ala Ala Ala Leu Ala260 265 270 Phe Ser Pro Leu Gly Leu Thr Thr Ser Pro Thr Thr Ser Ser LeuLeu 275 280 285 Gly Glu Ser Pro Ser Leu Pro Ser Pro Pro Asn Arg Ser ThrAla Ser 290 295 300 Gly Glu Gly Thr Cys Ala Val Cys Gly Asp Asn Ala AlaCys Gln His 305 310 315 320 Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys GlyPhe Phe Lys Arg Thr 325 330 335 Val Gln Lys Asn Ala Lys Tyr Val Cys LeuAla Asn Lys Asn Cys Pro 340 345 350 Val Asp Lys Arg Arg Arg Asn Arg CysGln Tyr Cys Arg Phe Gln Lys 355 360 365 Cys Leu Ser Val Gly Met Val LysGlu Val Val Arg Thr Asp Ser Leu 370 375 380 Lys Gly Arg Arg Gly Arg LeuPro Ser Lys Pro Lys Ser Pro Leu Gln 385 390 395 400 Gln Glu Pro Ser GlnPro Ser Pro Pro Ser Pro Pro Val Cys Met Met 405 410 415 Asn Ala Leu ValArg Ala Leu Thr Asp Ser Thr Pro Arg Asp Leu Asp 420 425 430 Tyr Ser ArgGly His Ala Cys Gly Ile Trp Lys Phe Pro Gly 435 440 445 17 643 PRT Susscrofa 17 Met Pro Cys Val Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly SerSer 1 5 10 15 Tyr Ala Ala Gln Thr Tyr Gly Ser Glu Tyr Thr Thr Glu IleMet Asn 20 25 30 Pro Asp Tyr Thr Lys Leu Thr Met Asp Leu Gly Ser Thr GluIle Thr 35 40 45 Ala Thr Ala Thr Thr Ser Leu Pro Ser Phe Ser Thr Phe MetGlu Gly 50 55 60 Tyr Ser Ser Asn Tyr Glu Leu Lys Pro Ser Cys Leu Tyr GlnMet Gln 65 70 75 80 Pro Ser Gly Pro Arg Pro Leu Ile Lys Met Glu Glu GlyArg Ala His 85 90 95 Gly Tyr His His His His His Asp His His His His HisHis His Gln 100 105 110 Gln Gln Gln Gln Gln Gln Gln Pro Pro Pro Gln GlnGln Gln Pro Ser 115 120 125 Ile Pro Pro Pro Ser Gly Pro Glu Asp Glu ValLeu Pro Ser Thr Ser 130 135 140 Met Tyr Phe Lys Gln Ser Pro Pro Ser ThrPro Thr Thr Pro Val Phe 145 150 155 160 Pro Gln Gln Ala Gly Ala Leu TrpGlu Asp Ala Leu Pro Ser Ala Gln 165 170 175 Gly Cys Ile Ala Pro Gly ProLeu Leu Asp Pro Pro Met Lys Ala Val 180 185 190 Pro Thr Val Ala Gly AlaArg Phe Pro Leu Phe His Phe Lys Pro Ser 195 200 205 Pro Pro His Pro ProAla Pro Ser Pro Ala Gly Gly His His Leu Gly 210 215 220 Tyr Asp Pro ThrAla Ala Ala Ala Leu Gly Leu Pro Leu Gly Ala Ala 225 230 235 240 Ala AlaAla Ala Ala Ala Ala Ala Gly Ser Gln Ala Ala Ala Leu Glu 245 250 255 GlyHis Pro Tyr Gly Leu Pro Leu Ala Lys Arg Ala Ala Ala Leu Ala 260 265 270Phe Ser Pro Leu Gly Leu Thr Thr Ser Pro Thr Thr Ser Ser Leu Leu 275 280285 Gly Glu Ser Pro Ser Leu Pro Ser Pro Pro Asn Arg Ser Thr Ala Ser 290295 300 Gly Glu Gly Thr Cys Ala Val Cys Gly Asp Asn Ala Ala Cys Gln His305 310 315 320 Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys Gly Phe Phe LysArg Thr 325 330 335 Val Gln Lys Asn Ala Lys Tyr Val Cys Leu Ala Asn LysAsn Cys Pro 340 345 350 Val Asp Lys Arg Arg Arg Asn Arg Cys Gln Tyr CysArg Phe Gln Lys 355 360 365 Cys Leu Ser Val Gly Met Val Lys Glu Val ValArg Thr Asp Ser Leu 370 375 380 Lys Gly Arg Arg Gly Arg Leu Pro Ser LysPro Lys Ser Pro Leu Gln 385 390 395 400 Gln Glu Pro Ser Gln Pro Ser ProPro Ser Pro Pro Val Cys Met Met 405 410 415 Asn Ala Leu Val Arg Ala LeuThr Asp Ser Thr Pro Arg Asp Leu Asp 420 425 430 Tyr Ser Arg Tyr Cys ProAla Asp Gln Ala Ala Ala Gly Thr Asp Ala 435 440 445 Glu His Val Gln GlnPhe Tyr Asn Leu Leu Thr Ala Ser Ile Asp Val 450 455 460 Ser Arg Ser TrpAla Glu Lys Ile Pro Gly Phe Thr Asp Leu Pro Lys 465 470 475 480 Glu AspGln Thr Leu Leu Ile Glu Ser Ala Phe Leu Glu Leu Phe Val 485 490 495 LeuArg Leu Ser Ile Arg Ser Asn Thr Ala Glu Asp Lys Phe Val Phe 500 505 510Cys Asn Gly Leu Val Leu His Arg Leu Gln Cys Leu Arg Gly Phe Gly 515 520525 Glu Trp Leu Asp Ser Ile Lys Asp Phe Ser Leu Arg Leu Gln Ser Leu 530535 540 Asn Leu Asp Ile Gln Ala Leu Ala Cys Leu Ser Ala Leu Ser Met Ile545 550 555 560 Thr Glu Arg His Gly Leu Lys Glu Pro Lys Arg Val Glu GluLeu Cys 565 570 575 Asn Lys Ile Thr Ser Ser Leu Lys Glu His Gln Ser LysGly Gln Ala 580 585 590 Leu Glu Pro Thr Glu Pro Lys Val Leu Arg Ala LeuVal Glu Leu Arg 595 600 605 Lys Ile Cys Thr Leu Gly Leu Gln Arg Ile PheTyr Leu Lys Leu Glu 610 615 620 Asp Leu Val Ser Pro Pro Ser Ile Ile AspLys Leu Phe Leu Asp Thr 625 630 635 640 Leu Pro Phe 18 143 PRT Homosapiens 18 Cys Gln His Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys Gly PhePhe 1 5 10 15 Lys Arg Thr Val Gln Lys Asn Ala Lys Tyr Val Cys Leu AlaAsn Lys 20 25 30 Asn Cys Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln TyrCys Arg 35 40 45 Phe Gln Lys Cys Leu Ser Val Gly Met Val Lys Glu Val ValArg Thr 50 55 60 Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys ProLys Ser 65 70 75 80 Pro Leu Gln Gln Glu Pro Ser Gln Pro Ser Pro Pro SerPro Pro Ile 85 90 95 Cys Met Met Asn Ala Leu Val Arg Ala Leu Thr Asp SerThr Pro Arg 100 105 110 Asp Leu Asp Tyr Ser Arg Val Ser Phe Met Ile SerCys Phe Gln Met 115 120 125 Asn Asp Gln Gly Leu Tyr Leu Trp Leu Leu ValIle Arg Val Asp 130 135 140 19 69 PRT Homo sapiens 19 Met Pro Cys ValGln Ala Gln Tyr Ser Pro Ser Pro Pro Gly Ser Ser 1 5 10 15 Tyr Ala AlaGln Thr Tyr Ser Ser Glu Tyr Thr Thr Glu Ile Met Asn 20 25 30 Pro Asp TyrThr Lys Leu Thr Met Asp Leu Gly Ser Thr Glu Ile Thr 35 40 45 Ala Thr AlaThr Thr Ser Leu Pro Ser Ile Ser Thr Phe Val Glu Gly 50 55 60 Tyr Ser SerAsn Tyr 65 20 1831 DNA Homo sapiens 20 gaattccggg ctcgctcagc tgctgcccagcctcggctgt gaggataggc tggctgggca 60 gcacgtctct ccccacaggg ctccctgagaccaccaggaa gagcccccaa ccaatcttgg 120 gattctccct tcgtgcggtt gtctgggacctttttccagg gtcaaagcag atcgtgagga 180 ggaagctgag atgccctgta tccaagcccaatatgggaca ccagcaccga gtccgggacc 240 ccgtgaccac ctggcaagcg accccctgacccctgagttc atcaagccca ccatggacct 300 ggccagcccc gaggcagccc ccgctgcccccactgccctg cccagcttca gcaccttcat 360 ggacggctac acaggagagt ttgacaccttcctctaccag ctgccaggaa cagtccagcc 420 atgctcctca gcctcctcct cggcctcctccacatcctcg tcctcagcca cctcccctgc 480 ctctgcctcc ttcaagttcg aggacttccaggtgtacggc tgctaccccg gccccctgag 540 cggcccagtg gatgaggccc tgtcctccagtggctctgac tactatggca gcccctgctc 600 ggccccgtcg ccctccacgc ccagcttccagccgccccag ctctctccct gggatggctc 660 cttcggccac ttctcgccca gccagacttacgaaggcctg cgggcatgga cagagcagct 720 gcccaaagcc tctgggcccc cacagcctccagccttcttt tccttcagtc ctcccaccgg 780 ccccagcccc agcctggccc agagccccctgaagttgttc ccctcacagg ccacccacca 840 gctgggggag ggagagagct attccatgcctacggccttc ccaggtttgg cacccacttc 900 tccacacctt gagggctcgg ggatactggatacacccgtg acctcaacca aggcccggag 960 cggggcccca ggtggaagtg aaggccgctgtgctgtgtgt ggggacaacg cttcatgcca 1020 gcattatggt gtccgcacat gtgagggctgcaagggcttc ttcaaggtac cgcgcagccc 1080 caggtggggc cttttgttgg aaatggagagaggctggcct catcccattg ggacctgtgg 1140 tctccccctg ggttctcctc ctagctaagtcctgtcctgc agggtgggat cagccctgcc 1200 aggtgggccg ccttcctgga gacccgtagatgccagggct ggaagctttc atttgccggg 1260 acactcgggc ccatgggatt gcacagagctggagggaggg gtgagatagg ggcagatagg 1320 agctgcaggg gtgcctggcg agcctctggttttcctctgc tcctctgcct gtcctctccc 1380 aactcaaggt tctagtggga agggtgcccccaggctctca tgttcctggc gtgagatgaa 1440 aggatcctgc ggagggtttg gttcttgagggctgggggtg gacttgggaa caggctgtgt 1500 gtttgtccca gcgatggtgc ctgcttagcttcccgtcccc accccccagc cccttggccc 1560 tctcctgtct gccctaggga gaaggcaggtggacaagggc ccatgaaaaa atacaggtgt 1620 ctagactgcc agggagaccc tggccccccagtagtgtgtc ctggggactt cctcagagcg 1680 agaaacctcc ccccaatgtc ttcaagacttttctctcccc cccccccaac cccgtctctc 1740 cctcccttgc cacccaaatg ttagaaaaatagctgtgaac agagagcgct tttgtctgca 1800 atggcagcag gatctggacg gtccggaatt c1831 21 250 DNA Homo sapiens 21 acacgggctc aaggaaccca agagagtggaagaactgcaa aacaagattg ttaattgtct 60 caaagaccac gtgactttca tcaatggggggttgaaccgc cccaattatt tgtgcaaact 120 gttggggaag ctcccataac ttcgtaccctttgcacacat gggctgcagc gcattttcta 180 cctgaaattg gaagacttgg tgccaccgccatcgatattt gacaaacttt tcctgtacac 240 tttacctttc 250 22 442 DNA ZebraFish 22 ggaacttcaa aacaagctca taaactgtct gaaggatcag gtgtcctgcagtggtgaatt 60 gtctaaactg ttggagaagc tgccggaggt gcgcgcgctg tgcacgcagggtctgcagcg 120 catcttttac ttgaaactgg aggatttggt gcccacgcct gcgatcattgataaactctt 180 tcatgacact ttaccattct aaacaatctc gtctgaactg aagcaggttctcgaaacctt 240 gttattcttc gatgttgcgg actttttagg aaatgaaaat ggcggatggtatttttaaag 300 attggagaaa gactgcccgg gacaaaactg aacagtccta tttggaggaaataattattc 360 aagaaaaaaa tattatggag gaaaagggac tattcttatg cctgtttaaagtgtattcag 420 ttatatactg aaaataaatg cg 442 23 492 DNA Mus musculus 23acaaaattgt aaattgtctt aaagaccatg tgactttcaa taatgggggt ttgaaccgac 60ccaactacct gtctaaactg ttggggaagc tgccagaact ccgcaccctt tgcacacagg 120gcctccagcg cattttctac ctgaaattgg aagacttggt accaccacca gcaataattg 180acaaactttt cctggacacc ttacctttct aagaccttct cccaagcacg tcaaagaact 240ggaaagaaaa aaaaaataac atccagaggg ggctggtcac atgggcagag agctggttga 300agtgtccagt tcaccttatc tcccttctgt agacccctag ccctcacccc ttaagtaaac 360aaacaaacaa acaaaccaca aataaaaact gtagctattt cctaacctgc aggcagaacc 420tgaaagggca ttttggctcc ggggcatcct ggatttagaa aacggacagc acacagtaca 480gtggtataaa ct 492 24 500 DNA Homo sapiens modified_base (420) N = A, C,G and/or T/U 24 agaactgcaa aacaagattg taaattgtct caaagaccac gtgactttcaacaatggggg 60 gttgaaccgc cccaattatt tgtccaaact gttggggaag ctcccagaacttcgtaccct 120 ttgcacacag gggctacagc gcattttcta cctgaaattg gaagacttggtgccaccgcc 180 agcaataatt gacaaacttt tcctggacac tttacctttc taagacctcctcccaagcac 240 ttcaaaggaa ctggaatgat aatggaaact gtcaagaggg ggcaagtcacatgggcagag 300 atagccgtgt gagcagtctc agctcaagct gccccccatt tctgtaaccctcctagcccc 360 cttgatccct aaagaaaaca aacaaacaaa caaaaactgt tgctatttcctaacctgcan 420 gcagaacctg aaagggcatt ttggctccgg ggcatcctgg atttagaacatggactacac 480 acaatacagt ggtataaact 500 25 499 DNA Homo sapiensmodified_base (420) N = A, C, G and/or T/U 25 agaactgcaa aacaagattgtaaattgtct caaagaccac gtgactttca acaatggggg 60 gttgaaccgc cccaattatttgtccaaact gttggggaag ctcccagaac ttcgtaccct 120 ttgcacacag gggctacagcgcattttcta cctgaaattg gaagacttgg tgccaccgcc 180 agcaataatt gacaaacttttcctggacac tttacctttc taagacctcc tcccaagcac 240 ttcaaaggaa ctggaatgataatggaaact gtcaagaggg ggcaagtcac atgggcagag 300 atagccgtgt gagcagtctcagctcaagct gccccccatt tctgtaaccc tcctagcccc 360 cttgatccct aaagaaaacaaacaaacaaa caaaaactgt tgctatttcc taacctgcan 420 gcagaacctg aaagggcattttggctccgg ggcatcctgg atttagaaca tggactacac 480 acaatacagt ggtataaac 49926 520 DNA Mus musculus 26 acaaaattgt aaattgtctt aaagaccatg tgactttcaataatgggggt ttgaaccgac 60 ccaactacct gtctaaactg ttggggaagc tgccagaactccgcaccctt tgcacacagg 120 gcctccagcg cattttctac ctgaaattgg aagacttggtaccaccacca gcaataattg 180 acaaactttt cctggacacc ttacctttct aagaccttctcccaagcacg tcaaagaact 240 ggaaagaaaa aaaaaataac atccagaggg ggctggtcacatgggcagag agctggttga 300 agtgtccagt tcaccttatc tcccttctgt agacccctagccctcacccc ttaagtaaac 360 aaacaaacaa acaaaccaca aataaaaact gtagctatttcctaacctgc aggcagaacc 420 tgaaagggca ttttggctcc ggggcatcct ggatttagaaaacggacagc acacagtaca 480 gtggtataaa ctttttatta tcagttcaaa atcagtttgt520 27 396 DNA Homo sapiens 27 tgaatactaa catgtcaatt gttttgtggagataagagtg aacgtttccc agggctggat 60 ggcactgtat ttagtctgta tggaaatggcaatttacata tttaaagcag cgacctcgta 120 gcaccatccc taattgaatt aattgccccggaacatctaa tttccttact ggtcagagag 180 aggtttaatt gttataaaaa cctggctcccctattagaaa cggggttagc aatttcacgg 240 gttatatatt ttagagaacc tcattaagtgctttttaaaa tgaaattcca gttccaggcg 300 aaccctgact atcaaatgag tggagatgacacccagcata tccagcaatt ctatgatctc 360 ctgactggct ccatggagat catccggggctgggca 396 28 2481 DNA Homo sapiens 28 cgaacttggg gggagtgcac agaagaacttcgggagcgca cgcgggacca gggaccaggc 60 tgagactcgg ggcgccagtc cgggcaggggcagcgggagc cggccggaga tgccctgtat 120 ccaagcccaa tatgggacac cagcaccgagtccgggaccc cgtgaccacc tggcaagcga 180 ccccctgacc cctgagttca tcaagcccaccatggacctg gccagccccg aggcagcccc 240 cgctgccccc actgccctgc ccagcttcagcaccttcatg gacggctaca caggagagtt 300 tgacaccttc ctctaccagc tgccaggaacagtccagcca tgctcctcag cctcctcctc 360 ggcctcctcc acatcctcgt cctcagccacctcccctgcc tctgcttcct tcaagttcga 420 ggacttccag gtgtacggct gctaccccggccccctgagc ggcccagtgg atgaggccct 480 gtcctccagt ggctctgact actatggcagcccctgctcg gccccgtcgc cctccacgcc 540 cagcttccag ccgccccagc tctctccctgggatggctcc ttcggccact tctcgcccag 600 ccagacttac gaaggcctgc gggcatggacagagcagctg cccaaagcct ctgggccccc 660 acagcctcca gccttctttt ccttcagtcctcccaccggc cccagcccca gcctggccca 720 gagccccctg aagttgttcc cctcacaggccacccaccag ctgggggagg gagagagcta 780 ttccatgcct acggccttcc caggtttggcacccacttct ccacaccttg agggctcggg 840 gatactggat acacccgtga cctcaaccaaggcccggagc ggggccccag gtggaagtga 900 aggccgctgt gctgtgtgtg gggacaacgcttcatgccag cattatggtg tccgcacatg 960 tgagggctgc aagggcttct tcaagcgcacagtgcagaaa aacgccaagt acatctgcct 1020 ggctaacaag gactgccctg tggacaagaggcggcgaaac cgctgccagt tctgccgctt 1080 ccagaagtgc ctggcggtgg gcatggtgaaggaagttgtc cgaacagaca gcctgaaggg 1140 gcggcggggc cggctacctt caaaacccaagcagccccca gatgcctccc ctgccaatct 1200 cctcacttcc ctggtccgtg cacacctggactcagggccc agcactgcca aactggacta 1260 ctccaagttc caggagctgg tgctgccccactttgggaag gaagatgctg gggatgtaca 1320 gcagttctac gacctgctct ccggttctctggaggtcatc cgcaagtggg cggagaagat 1380 ccctggcttt gctgagctgt caccggctgaccaggacctg ttgctggagt cggccttcct 1440 ggagctcttc atcctccgcc tggcgtacaggtctaagcca ggcgagggca agctcatctt 1500 ctgctcaggc ctggtgctac accggctgcagtgtgcccgt ggcttcgggg actggattga 1560 cagtatcctg gccttctcaa ggtccctgcacagcttgctt gtcgatgtcc ctgccttcgc 1620 ctgcctctct gcccttgtcc tcatcaccgaccggcatggg ctgcaggagc cgcggcgggt 1680 ggaggagctg cagaaccgca tcgccagctgcctgaaggag cacgtggcag ctgtggcggg 1740 cgagccccag ccagccagct gcctgtcacgtctgttgggc aaactgcccg agctgcggac 1800 cctgtgcacc cagggcctgc agcgcatcttctacctcaag ctggaggact tggtgccccc 1860 tccacccatc attgacaaga tcttcatggacacgctgccc ttctgacccc tgcctgcctg 1920 ggaacacgtg tgcacatgcg cactctctcatatgccaccc catgtgcctt tagtccacgg 1980 accccagagc acccccaagc ctgggcttagctgcagaaca gagggacctg ctcacctgcc 2040 caaaggggat gaagggaggg aggctcaaggcccttggggg agggggatgc cttcatgggg 2100 gtgacccacg atgtgttctt atcccccccgcctggccacc ggcctttatg ttttttgtaa 2160 gataaaccgt ttttaacaca tagcgccgtgctgtaaataa gcccagtact gctgtaaata 2220 caggaagaaa gagcttgagg tgggagcgggctgggaggaa gggatgggcc ccggccttcc 2280 tgggcagcct ttccagcctc ctgctgggctctctcttcct accctccttc cacatgtaca 2340 tgtacataaa ctgtcactct aggaagaagacaaatgacag attctgacca tttatatttg 2400 tgtattttcc aggatttata gtatgtgacttttctgatta atatatttaa tatattgaat 2460 aaaaaataga catgtagttg g 2481 292469 DNA Homo sapiens 29 ttcggcacga gtgcacttcg gcagagttga atgaatgaagagagacgcgg agaactccta 60 aggaggagat tggacaggct ggactcccca ttgcttttctaaaaatcttg gaaactttgt 120 ccttcattga attacgacac tgtccacctt taatttcctcgaaaacgcct gtaactcggc 180 tgaagcttca gtacctttat ggacaactac agcacaggctacgacgtcaa gccaccttgc 240 ttgtaccaaa tgcccctgtc cggacagcag tcctccattaaggtagaaga cattcagatg 300 cacaactacc agcaacacag ccacctgccc ccccagtctgaggagatgat gccgcactcc 360 gggtcggttt actacaagcc ctcctcgccc ccgacgcccaccaccccggg cttccaggtg 420 cagcacagcc ccatgtggga cgacccggga tctctccacaacttccacca gaactacgtg 480 gccactacgc acatgatcga gcagaggaaa acgccagtctcccgcctctc cctcttctcc 540 tttaagcaat cgccccctgg caccccggtg tctagttgccagatgcgctt cgacgggccc 600 ctgcacgtcc ccatgaaccc ggagcccgcc ggcagccaccacgtggtgga cgggcagacc 660 ttcgctgtgc ccaaccccat tcgcaagccc gcgtccatgggcttcccggg cctgcagatc 720 ggccacgcgt ctcagctgct cgacacgcag gtgccctcaccgccgtcgcg gggctccccc 780 tccaacgagg ggctctgcgc tgtgtgtggg gacaacgcggcctgccaaca ctacggcgtg 840 cgcacctgtg agggctgcaa aggcttcttt aagcgcacagtgcaaaaaaa tgcaaaatac 900 gtgtgtttag caaataaaaa ctgcccagtg gacaagcgtcgccggaatcg ctgtcagtac 960 tgccgatttc agaagtgcct ggctgttggg atggtcaaagaagtggttcg cacagacagt 1020 ttaaaaggcc ggagaggtcg tttgccctcg aaaccgaagagcccacagga gccctctccc 1080 ccttcgcccc cggtgagtct gatcagtgcc ctcgtcagggcccatgtcga ctccaacccg 1140 gctatgacca gcctggacta ttccaggttc caggcgaaccctgactatca aatgagtgga 1200 gatgacaccc agcatatcca gcaattctat gatctcctgactggctccat ggagatcatc 1260 cggggctggg cagagaagat ccctggcttc gcagacctgcccaaagccga ccaagacctg 1320 ctttttgaat cagctttctt agaactgttt gtccttcgattagcatacag gtccaaccca 1380 gtggagggta aactcatctt ttggaatggg tgggtcttgcacaggttgca atgcgttcgt 1440 ggctttgggg aatggattga ttccattgtt gaattctcctccaacttgca gaatatgaac 1500 atcgacattt ctgccttctc ctgcattgct gccctggctatggtcacaga gagacacggg 1560 ctcaaggaac ccaagagagt ggaagaactc caaaacaagattgtaaattg tctcaaagac 1620 cacgtgactt tcaacaatgg ggggttgaac cgccccaattatttgtccaa actgttgggg 1680 aagctcccag aacttcgtac cctttgcaca caggggctacagcgcatttt ctacctgaaa 1740 ttggaagact tggtgccacc gccagcaata attgacaaacttttcctgga cactttacct 1800 ttctaagacc tcctcccaag cacttcaaag gaactggaatgataatggaa actgtcaaga 1860 gggggcaagt cacatgggca gagatagccg tgtgagcagtctcagctcaa gctgcccccc 1920 atttctgtaa ccctcctagc ccccttgatc cctaaagaaaacaaacaaac aaacaaaaac 1980 tgttgctatt tcctaacctg caggcagaac ctgaaagggcattttggctc cggggcatcc 2040 tggatttaga acatggacta cacacaatac agtggtataaactttttatt ctcagtttaa 2100 aaatcagttt gttgttcaga agaaagattg ctataatgtataatgggaaa tgtttgccca 2160 tgcttggttg ttgcagttca gacaaatgtg acacacacacacatacacac acacacacac 2220 acacagagac acatcttaag gggacccaca agtattgccctttaacaaga cttcaaagtt 2280 ttctgctgta aagaaagctg taatatatag taaaactaaatgttgcgtgg gtggcatgag 2340 ttgaagaagg ccaaggcttg taaatttacc caatgcagtttggcttttta aattattttg 2400 tgcctattta tgaataaata ttaccaattc taaaagataagtgtgtttcc cacaaaaaaa 2460 aaaaaaaaa 2469 30 598 PRT Homo sapiens 30 MetPro Cys Ile Gln Ala Gln Tyr Gly Thr Pro Ala Pro Ser Pro Gly 1 5 10 15Pro Arg Asp His Leu Ala Ser Asp Pro Leu Thr Pro Glu Phe Ile Lys 20 25 30Pro Thr Met Asp Leu Ala Ser Pro Glu Ala Ala Pro Ala Ala Pro Thr 35 40 45Ala Leu Pro Ser Phe Ser Thr Phe Met Asp Gly Tyr Thr Gly Glu Phe 50 55 60Asp Thr Phe Leu Tyr Gln Leu Pro Gly Thr Val Gln Pro Cys Ser Ser 65 70 7580 Ala Ser Ser Ser Ala Ser Ser Thr Ser Ser Ser Ser Ala Thr Ser Pro 85 9095 Ala Ser Ala Ser Phe Lys Phe Glu Asp Phe Gln Val Tyr Gly Cys Tyr 100105 110 Pro Gly Pro Leu Ser Gly Pro Val Asp Glu Ala Leu Ser Ser Ser Gly115 120 125 Ser Asp Tyr Tyr Gly Ser Pro Cys Ser Ala Pro Ser Pro Ser ThrPro 130 135 140 Ser Phe Gln Pro Pro Gln Leu Ser Pro Trp Asp Gly Ser PheGly His 145 150 155 160 Phe Ser Pro Ser Gln Thr Tyr Glu Gly Leu Arg AlaTrp Thr Glu Gln 165 170 175 Leu Pro Lys Ala Ser Gly Pro Pro Gln Pro ProAla Phe Phe Ser Phe 180 185 190 Ser Pro Pro Thr Gly Pro Ser Pro Ser LeuAla Gln Ser Pro Leu Lys 195 200 205 Leu Phe Pro Ser Gln Ala Thr His GlnLeu Gly Glu Gly Glu Ser Tyr 210 215 220 Ser Met Pro Thr Ala Phe Pro GlyLeu Ala Pro Thr Ser Pro His Leu 225 230 235 240 Glu Gly Ser Gly Ile LeuAsp Thr Pro Val Thr Ser Thr Lys Ala Arg 245 250 255 Ser Gly Ala Pro GlyGly Ser Glu Gly Arg Cys Ala Val Cys Gly Asp 260 265 270 Asn Ala Ser CysGln His Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys 275 280 285 Gly Phe PheLys Arg Thr Val Gln Lys Asn Ala Lys Tyr Ile Cys Leu 290 295 300 Ala AsnLys Asp Cys Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln 305 310 315 320Phe Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Val Lys Glu Val 325 330335 Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys 340345 350 Pro Lys Gln Pro Pro Asp Ala Ser Pro Ala Asn Leu Leu Thr Ser Leu355 360 365 Val Arg Ala His Leu Asp Ser Gly Pro Ser Thr Ala Lys Leu AspTyr 370 375 380 Ser Lys Phe Gln Glu Leu Val Leu Pro His Phe Gly Lys GluAsp Ala 385 390 395 400 Gly Asp Val Gln Gln Phe Tyr Asp Leu Leu Ser GlySer Leu Glu Val 405 410 415 Ile Arg Lys Trp Ala Glu Lys Ile Pro Gly PheAla Glu Leu Ser Pro 420 425 430 Ala Asp Gln Asp Leu Leu Leu Glu Ser AlaPhe Leu Glu Leu Phe Ile 435 440 445 Leu Arg Leu Ala Tyr Arg Ser Lys ProGly Glu Gly Lys Leu Ile Phe 450 455 460 Cys Ser Gly Leu Val Leu His ArgLeu Gln Cys Ala Arg Gly Phe Gly 465 470 475 480 Asp Trp Ile Asp Ser IleLeu Ala Phe Ser Arg Ser Leu His Ser Leu 485 490 495 Leu Val Asp Val ProAla Phe Ala Cys Leu Ser Ala Leu Val Leu Ile 500 505 510 Thr Asp Arg HisGly Leu Gln Glu Pro Arg Arg Val Glu Glu Leu Gln 515 520 525 Asn Arg IleAla Ser Cys Leu Lys Glu His Val Ala Ala Val Ala Gly 530 535 540 Glu ProGln Pro Ala Ser Cys Leu Ser Arg Leu Leu Gly Lys Leu Pro 545 550 555 560Glu Leu Arg Thr Leu Cys Thr Gln Gly Leu Gln Arg Ile Phe Tyr Leu 565 570575 Lys Leu Glu Asp Leu Val Pro Pro Pro Pro Ile Ile Asp Lys Ile Phe 580585 590 Met Asp Thr Leu Pro Phe 595 31 601 PRT Mus musculus 31 Met ProCys Ile Gln Ala Gln Tyr Gly Thr Pro Ala Thr Ser Pro Gly 1 5 10 15 ProArg Asp His Leu Thr Gly Asp Pro Leu Ala Leu Glu Phe Gly Lys 20 25 30 ProThr Met Asp Leu Ala Ser Pro Glu Thr Ala Pro Ala Ala Pro Ala 35 40 45 ThrLeu Pro Ser Phe Ser Thr Phe Met Asp Gly Tyr Thr Gly Glu Phe 50 55 60 AspThr Phe Leu Tyr Gln Leu Pro Gly Thr Thr Gln Pro Cys Ser Ser 65 70 75 80Ala Cys Ser Ser Ala Ser Ser Thr Ser Ser Ser Ser Ser Ser Ala Thr 85 90 95Ser Pro Ala Ser Ala Ser Phe Lys Phe Glu Asp Phe Gln Val Tyr Gly 100 105110 Cys Tyr Pro Gly Thr Leu Ser Gly Pro Leu Asp Glu Thr Leu Ser Ser 115120 125 Ser Gly Ser Glu Tyr Tyr Gly Ser Pro Cys Ser Ala Pro Ser Pro Ser130 135 140 Thr Pro Asn Phe Gln Pro Ser Gln Leu Ser Pro Trp Asp Gly SerPhe 145 150 155 160 Gly His Phe Ser Pro Ser Gln Thr Tyr Glu Gly Leu TrpAla Trp Thr 165 170 175 Glu Gln Leu Pro Lys Ala Ser Ser Gly Pro Pro ProPro Pro Thr Phe 180 185 190 Phe Ser Phe Ser Pro Pro Thr Gly Pro Ser ProSer Leu Ala Gln Ser 195 200 205 Ser Leu Lys Leu Phe Pro Pro Pro Ala ThrHis Gln Leu Gly Glu Gly 210 215 220 Glu Ser Tyr Ser Met Pro Ala Ala PhePro Gly Leu Ala Pro Thr Ser 225 230 235 240 Pro Asn Arg Asp Thr Ser GlyIle Leu Asp Ala Pro Val Thr Ser Thr 245 250 255 Lys Ser Arg Ser Gly AlaSer Gly Gly Ser Glu Gly Arg Cys Ala Val 260 265 270 Cys Gly Asp Asn AlaSer Cys Gln His Tyr Gly Val Arg Thr Cys Glu 275 280 285 Gly Cys Lys GlyPhe Phe Lys Arg Thr Val Gln Lys Ser Ala Lys Tyr 290 295 300 Ile Cys LeuAla Asn Lys Asp Cys Pro Val Asp Lys Arg Arg Arg Asn 305 310 315 320 ArgCys Gln Phe Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Val 325 330 335Lys Glu Val Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu 340 345350 Pro Ser Lys Pro Lys Gln Pro Pro Asp Ala Ser Pro Thr Asn Leu Leu 355360 365 Thr Ser Leu Ile Arg Ala His Leu Asp Ser Gly Pro Ser Thr Ala Lys370 375 380 Leu Asp Tyr Ser Lys Phe Gln Glu Leu Val Leu Pro Arg Phe GlyLys 385 390 395 400 Glu Asp Ala Gly Asp Val Gln Gln Phe Tyr Asp Leu LeuSer Gly Ser 405 410 415 Leu Asp Val Ile Arg Lys Trp Ala Glu Lys Ile ProGly Phe Ile Glu 420 425 430 Leu Cys Pro Gly Asp Gln Asp Leu Leu Leu GluSer Ala Phe Leu Glu 435 440 445 Leu Phe Ile Leu Arg Leu Ala Tyr Arg SerLys Pro Gly Glu Gly Lys 450 455 460 Leu Ile Phe Cys Ser Gly Leu Val LeuHis Gln Leu Gln Cys Ala Arg 465 470 475 480 Gly Phe Gly Asp Trp Ile AspAsn Ile Leu Ala Phe Ser Arg Ser Leu 485 490 495 His Ser Leu Gly Val AspVal Pro Ala Phe Ala Cys Leu Ser Ala Leu 500 505 510 Val Leu Ile Thr AspArg His Gly Leu Gln Asp Pro Arg Arg Val Glu 515 520 525 Glu Leu Gln AsnArg Ile Ala Ser Cys Leu Lys Glu His Met Ala Thr 530 535 540 Val Ala GlyAsp Pro Gln Pro Ala Ser Cys Leu Ser Arg Leu Leu Gly 545 550 555 560 LysLeu Pro Glu Leu Arg Thr Leu Cys Thr Gln Gly Leu Gln Arg Ile 565 570 575Phe Cys Leu Lys Leu Glu Asp Leu Val Pro Pro Pro Pro Ile Val Asp 580 585590 Lys Ile Phe Met Asp Thr Leu Ser Phe 595 600 32 597 PRT RAT 32 MetPro Cys Ile Gln Ala Gln Tyr Gly Thr Pro Ala Thr Ser Pro Gly 1 5 10 15Pro Arg Asp His Leu Thr Gly Asp Pro Leu Ala Leu Glu Phe Ser Lys 20 25 30Pro Thr Met Asp Leu Ala Ser Pro Glu Thr Ala Pro Thr Ala Pro Ala 35 40 45Thr Leu Pro Ser Phe Ser Thr Phe Met Asp Gly Gly Tyr Thr Gly Glu 50 55 60Phe Asp Thr Phe Leu Tyr Gln Leu Pro Gly Thr Ala Gln Pro Cys Ser 65 70 7580 Ser Ala Ser Ser Thr Ser Ser Ser Ser Ser Ser Ala Thr Ser Pro Ala 85 9095 Ser Ala Ser Phe Lys Phe Glu Asp Phe Gln Val Tyr Gly Cys Tyr Pro 100105 110 Gly Thr Leu Ser Gly Pro Leu Asp Glu Thr Leu Ser Ser Ser Gly Ser115 120 125 Asp Tyr Tyr Gly Ser Pro Cys Ser Ala Pro Ser Pro Pro Thr ProAsn 130 135 140 Phe Gln Pro Ser Gln Leu Ser Pro Trp Asp Gly Ser Phe GlyHis Phe 145 150 155 160 Ser Pro Ser Gln Thr Tyr Glu Gly Leu Arg Val TrpThr Glu Gln Leu 165 170 175 Pro Lys Ala Ser Gly Pro Pro Pro Pro Pro ThrPhe Phe Ser Phe Ser 180 185 190 Pro Pro Thr Gly Pro Ser Pro Ser Leu AlaGln Ser Ser Leu Lys Leu 195 200 205 Phe Pro Ala Pro Ala Thr His Gln LeuGly Glu Gly Glu Ser Tyr Ser 210 215 220 Val Pro Ala Ala Phe Pro Gly LeuAla Pro Thr Ser Pro Asn Cys Asp 225 230 235 240 Thr Ser Gly Ile Leu AspAla Pro Val Thr Ser Thr Lys Ala Arg Ser 245 250 255 Gly Ser Ser Gly GlySer Glu Gly Arg Cys Ala Val Cys Gly Asp Asn 260 265 270 Ala Ser Cys GlnHis Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys Gly 275 280 285 Phe Phe LysArg Thr Val Gln Lys Ser Ala Lys Tyr Ile Cys Leu Ala 290 295 300 Asn LysAsp Cys Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln Phe 305 310 315 320Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Val Lys Glu Val Val 325 330335 Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro 340345 350 Lys Gln Pro Pro Asp Ala Ser Pro Thr Asn Leu Leu Thr Ser Leu Ile355 360 365 Arg Ala His Leu Asp Ser Gly Pro Asn Thr Ala Lys Leu Asp TyrSer 370 375 380 Lys Phe Gln Glu Leu Val Leu Pro Arg Phe Gly Lys Glu AspAla Gly 385 390 395 400 Asp Val Gln Gln Phe Tyr Asp Leu Leu Ser Gly SerLeu Asp Val Ile 405 410 415 Arg Lys Trp Ala Glu Lys Ile Pro Gly Phe IleGlu Leu Ser Pro Gly 420 425 430 Asp Gln Asp Leu Leu Leu Glu Ser Ala PheLeu Glu Leu Phe Ile Leu 435 440 445 Arg Leu Ala Tyr Arg Ser Lys Pro GlyGlu Gly Lys Leu Ile Phe Cys 450 455 460 Ser Gly Leu Val Leu His Arg LeuGln Cys Ala Arg Gly Phe Gly Asp 465 470 475 480 Trp Ile Asp Asn Ile LeuAla Phe Ser Arg Ser Leu His Ser Leu Gly 485 490 495 Val Asp Val Pro AlaPhe Ala Cys Leu Ser Ala Leu Val Leu Ile Thr 500 505 510 Asp Arg His GlyLeu Gln Asp Pro Arg Arg Val Glu Glu Leu Gln Asn 515 520 525 Arg Ile AlaSer Cys Leu Lys Glu His Met Ala Ala Val Ala Gly Asp 530 535 540 Pro GlnPro Ala Ser Cys Leu Ser Arg Leu Leu Gly Lys Leu Pro Glu 545 550 555 560Leu Arg Thr Leu Cys Thr Gln Gly Leu Gln Arg Ile Phe Cys Leu Lys 565 570575 Leu Glu Asp Leu Val Pro Pro Pro Pro Ile Val Asp Lys Ile Phe Met 580585 590 Asp Thr Leu Ser Phe 595 33 535 PRT Homo sapiens 33 Met Asp AsnTyr Ser Thr Gly Tyr Asp Val Lys Pro Pro Cys Leu Tyr 1 5 10 15 Gln MetPro Leu Ser Gly Gln Gln Ser Ser Ile Lys Val Glu Asp Ile 20 25 30 Gln MetHis Asn Tyr Gln Gln His Ser His Leu Pro Pro Gln Ser Glu 35 40 45 Glu MetMet Pro His Ser Gly Ser Val Tyr Tyr Lys Pro Ser Ser Pro 50 55 60 Pro ThrPro Thr Thr Pro Gly Phe Gln Val Gln His Ser Pro Met Trp 65 70 75 80 AspAsp Pro Gly Ser Leu His Asn Phe His Gln Asn Tyr Val Ala Thr 85 90 95 ThrHis Met Ile Glu Gln Arg Lys Thr Pro Val Ser Arg Leu Ser Leu 100 105 110Phe Ser Phe Lys Gln Ser Pro Pro Gly Thr Pro Val Ser Ser Cys Gln 115 120125 Met Arg Phe Asp Gly Pro Leu His Val Pro Met Asn Pro Glu Pro Ala 130135 140 Gly Ser His His Val Val Asp Gly Gln Thr Phe Ala Val Pro Asn Pro145 150 155 160 Ile Arg Lys Pro Ala Ser Met Gly Phe Pro Gly Leu Gln IleGly His 165 170 175 Ala Ser Gln Leu Leu Asp Thr Gln Val Pro Ser Pro ProSer Arg Gly 180 185 190 Ser Pro Ser Asn Glu Gly Leu Cys Ala Val Cys GlyAsp Asn Ala Ala 195 200 205 Cys Gln His Tyr Gly Val Arg Thr Cys Glu GlyCys Lys Gly Phe Phe 210 215 220 Lys Arg Thr Val Gln Lys Asn Ala Lys TyrVal Cys Leu Ala Asn Lys 225 230 235 240 Asn Cys Pro Val Asp Lys Arg ArgArg Asn Arg Cys Gln Tyr Cys Arg 245 250 255 Phe Gln Lys Cys Leu Ala ValGly Met Val Lys Glu Val Val Arg Thr 260 265 270 Asp Ser Leu Lys Gly ArgArg Gly Arg Leu Pro Ser Lys Pro Lys Ser 275 280 285 Pro Gln Glu Pro SerPro Pro Ser Pro Pro Val Ser Leu Ile Ser Ala 290 295 300 Leu Val Arg AlaHis Val Asp Ser Asn Pro Ala Met Thr Ser Leu Asp 305 310 315 320 Tyr SerArg Phe Gln Ala Asn Pro Asp Tyr Gln Met Ser Gly Asp Asp 325 330 335 ThrGln His Ile Gln Gln Phe Tyr Asp Leu Leu Thr Gly Ser Met Glu 340 345 350Ile Ile Arg Gly Trp Ala Glu Lys Ile Pro Gly Phe Ala Asp Leu Pro 355 360365 Lys Ala Asp Gln Asp Leu Leu Phe Glu Ser Ala Phe Leu Glu Leu Phe 370375 380 Val Leu Arg Leu Ala Tyr Arg Ser Asn Pro Val Glu Gly Lys Leu Ile385 390 395 400 Phe Trp Asn Gly Trp Val Leu His Arg Leu Gln Cys Val ArgGly Phe 405 410 415 Gly Glu Trp Ile Asp Ser Ile Val Glu Phe Ser Ser AsnLeu Gln Asn 420 425 430 Met Asn Ile Asp Ile Ser Ala Phe Ser Cys Ile AlaAla Leu Ala Met 435 440 445 Val Thr Glu Arg His Gly Leu Lys Glu Pro LysArg Val Glu Glu Leu 450 455 460 Gln Asn Lys Ile Val Asn Cys Leu Lys AspHis Val Thr Phe Asn Asn 465 470 475 480 Gly Gly Leu Asn Arg Pro Asn TyrLeu Ser Lys Leu Leu Gly Lys Leu 485 490 495 Pro Glu Leu Arg Thr Leu CysThr Gln Gly Leu Gln Arg Ile Phe Tyr 500 505 510 Leu Lys Leu Glu Asp LeuVal Pro Pro Pro Ala Ile Ile Asp Lys Leu 515 520 525 Phe Leu Asp Thr LeuPro Phe 530 535 34 597 PRT RAT 34 Met Pro Cys Val Gln Ala Gln Tyr GlySer Ser Pro Gln Gly Ala Ser 1 5 10 15 Pro Ala Ser Gln Ser Tyr Ser TyrHis Ser Ser Gly Glu Tyr Ser Ser 20 25 30 Asp Phe Leu Thr Pro Glu Phe ValLys Phe Ser Met Asp Leu Thr Asn 35 40 45 Thr Glu Ile Thr Ala Thr Thr SerLeu Pro Ser Phe Ser Thr Phe Met 50 55 60 Asp Asn Tyr Ser Thr Gly Tyr AspVal Lys Pro Pro Cys Leu Tyr Gln 65 70 75 80 Met Pro Leu Ser Gly Gln GlnSer Ser Ile Lys Val Glu Asp Ile Gln 85 90 95 Met His Asn Tyr Gln Gln HisSer His Leu Pro Pro Gln Ser Glu Glu 100 105 110 Met Met Pro His Ser GlySer Val Tyr Tyr Lys Pro Ser Ser Pro Pro 115 120 125 Thr Pro Ser Thr ProGly Phe Gln Val Gln His Ser Pro Met Trp Asp 130 135 140 Asp Pro Gly SerLeu His Asn Phe His Gln Asn Tyr Val Ala Thr Thr 145 150 155 160 His MetIle Glu Gln Arg Lys Thr Pro Val Ser Arg Leu Ser Leu Phe 165 170 175 SerPhe Lys Gln Ser Ala Pro Gly Thr Pro Val Ser Ser Cys Gln Met 180 185 190Arg Phe Asp Gly Pro Leu His Val Pro Met Asn Pro Glu Pro Ala Gly 195 200205 Ser His His Val Val Asp Gly Gln Thr Phe Ala Val Pro Asn Pro Ile 210215 220 Arg Lys Pro Ala Ser Met Gly Phe Pro Gly Leu Gln Ile Gly His Ala225 230 235 240 Ser Gln Leu Leu Asp Thr Gln Val Pro Pro Ser Pro Ser ArgGly Ser 245 250 255 Pro Ser Asn Glu Gly Leu Cys Ala Val Cys Gly Asp AsnAla Ala Cys 260 265 270 Gln His Tyr Gly Val Arg Thr Cys Glu Gly Cys LysGly Phe Phe Lys 275 280 285 Arg Thr Val Gln Lys Asn Ala Lys Tyr Val CysLeu Ala Asn Lys Asn 290 295 300 Cys Pro Val Asp Lys Arg Arg Arg Asn ArgCys Gln Tyr Cys Arg Phe 305 310 315 320 Gln Lys Cys Leu Ala Val Gly MetVal Lys Glu Val Val Arg Thr Asp 325 330 335 Ser Leu Lys Gly Arg Arg GlyArg Leu Pro Ser Lys Pro Lys Ser Pro 340 345 350 Gln Asp Pro Ser Pro ProSer Pro Pro Gly Ser Asp Gln Cys Pro Arg 355 360 365 Gln Thr His Val AspSer Asn Pro Ala Met Thr Ser Leu Asp Tyr Ser 370 375 380 Arg Phe Gln AlaAsn Pro Asp Tyr Gln Met Ser Gly Asp Asp Thr Gln 385 390 395 400 His IleGln Gln Phe Tyr Asp Leu Leu Thr Gly Ser Met Glu Ile Ile 405 410 415 ArgGly Trp Ala Glu Lys Ile Pro Gly Phe Ala Asp Leu Pro Lys Ala 420 425 430Ser Gln Asp Leu Leu Phe Glu Ser Ala Phe Leu Glu Leu Phe Val Leu 435 440445 Arg Leu Ala Tyr Arg Ser Asn Pro Val Glu Gly Lys Leu Ile Phe Cys 450455 460 Asn Gly Val Val Leu His Arg Leu Gln Cys Val Arg Gly Phe Gly Glu465 470 475 480 Trp Ile Asp Ser Ile Val Glu Phe Ser Ser Asn Leu Gln AsnMet Asn 485 490 495 Ile Asp Ile Ser Ala Phe Ser Cys Ile Ala Ala Leu AlaMet Val Thr 500 505 510 Glu Arg His Gly Leu Lys Glu Pro Lys Arg Val GluGlu Leu Gln Asn 515 520 525 Lys Ile Val Asn Cys Leu Lys Asp His Val ThrPhe Asn Asn Gly Gly 530 535 540 Leu Asn Arg Pro Asn Tyr Leu Ser Lys LeuLeu Gly Lys Leu Pro Glu 545 550 555 560 Leu Arg Thr Leu Cys Thr Gln GlyLeu Gln Arg Ile Phe Tyr Leu Lys 565 570 575 Leu Glu Asp Leu Val Pro ProPro Ala Ile Ile Asp Lys Leu Phe Leu 580 585 590 Asp Thr Leu Pro Phe 59535 598 PRT Homo sapiens 35 Met Pro Cys Ile Gln Ala Gln Tyr Gly Thr ProAla Pro Ser Pro Gly 1 5 10 15 Pro Arg Asp His Leu Ala Ser Asp Pro LeuThr Pro Glu Phe Ile Lys 20 25 30 Pro Thr Met Asp Leu Ala Ser Pro Glu AlaAla Pro Ala Ala Pro Thr 35 40 45 Ala Leu Pro Ser Phe Ser Thr Phe Met AspGly Tyr Thr Gly Glu Phe 50 55 60 Asp Thr Phe Leu Tyr Gln Leu Pro Gly ThrVal Gln Pro Cys Ser Ser 65 70 75 80 Ala Ser Ser Ser Ala Ser Ser Thr SerSer Ser Ser Ala Thr Ser Pro 85 90 95 Ala Ser Ala Ser Phe Lys Phe Glu AspPhe Gln Val Tyr Gly Cys Tyr 100 105 110 Pro Gly Pro Leu Ser Gly Pro ValAsp Glu Ala Leu Ser Ser Ser Gly 115 120 125 Ser Asp Tyr Tyr Gly Ser ProCys Ser Ala Pro Ser Pro Ser Thr Pro 130 135 140 Ser Phe Gln Pro Pro GlnLeu Ser Pro Trp Asp Gly Ser Phe Gly His 145 150 155 160 Phe Ser Pro SerGln Thr Tyr Glu Gly Leu Arg Ala Trp Thr Glu Gln 165 170 175 Leu Pro LysAla Ser Gly Pro Pro Gln Pro Pro Ala Phe Phe Ser Phe 180 185 190 Ser ProPro Thr Gly Pro Ser Pro Ser Leu Ala Gln Ser Pro Leu Lys 195 200 205 LeuPhe Pro Ser Gln Ala Thr His Gln Leu Gly Glu Gly Glu Ser Tyr 210 215 220Ser Met Pro Thr Ala Phe Pro Gly Leu Ala Pro Thr Ser Pro His Leu 225 230235 240 Glu Gly Ser Gly Ile Leu Asp Thr Pro Val Thr Ser Thr Lys Ala Arg245 250 255 Ser Gly Ala Pro Gly Gly Ser Glu Gly Arg Cys Ala Val Cys GlyAsp 260 265 270 Asn Ala Ser Cys Gln His Tyr Gly Val Arg Thr Cys Glu GlyCys Lys 275 280 285 Gly Phe Phe Lys Arg Thr Val Gln Lys Asn Ala Lys TyrIle Cys Leu 290 295 300 Ala Asn Lys Asp Cys Pro Val Asp Lys Arg Arg ArgAsn Arg Cys Gln 305 310 315 320 Phe Cys Arg Phe Gln Lys Cys Leu Ala ValGly Met Val Lys Glu Val 325 330 335 Val Arg Thr Asp Ser Leu Lys Gly ArgArg Gly Arg Leu Pro Ser Lys 340 345 350 Pro Lys Gln Pro Pro Asp Ala SerPro Ala Asn Leu Leu Thr Ser Leu 355 360 365 Val Arg Ala His Leu Asp SerGly Pro Ser Thr Ala Lys Leu Asp Tyr 370 375 380 Ser Lys Phe Gln Glu LeuVal Leu Pro His Phe Gly Lys Glu Asp Ala 385 390 395 400 Gly Asp Val GlnGln Phe Tyr Asp Leu Leu Ser Gly Ser Leu Glu Val 405 410 415 Ile Arg LysTrp Ala Glu Lys Ile Pro Gly Phe Ala Glu Leu Ser Pro 420 425 430 Ala AspGln Asp Leu Leu Leu Glu Ser Ala Phe Leu Glu Leu Phe Ile 435 440 445 LeuArg Leu Ala Tyr Arg Ser Lys Pro Gly Glu Gly Lys Leu Ile Phe 450 455 460Cys Ser Gly Leu Val Leu His Arg Leu Gln Cys Ala Arg Gly Phe Gly 465 470475 480 Asp Trp Ile Asp Ser Ile Leu Ala Phe Ser Arg Ser Leu His Ser Leu485 490 495 Leu Val Asp Val Pro Ala Phe Ala Cys Leu Ser Ala Leu Val LeuIle 500 505 510 Thr Asp Arg His Gly Leu Gln Glu Pro Arg Arg Val Glu GluLeu Gln 515 520 525 Asn Arg Ile Ala Ser Cys Leu Lys Glu His Val Ala AlaVal Ala Gly 530 535 540 Glu Pro Gln Pro Ala Ser Cys Leu Ser Arg Leu LeuGly Lys Leu Pro 545 550 555 560 Glu Leu Arg Thr Leu Cys Thr Gln Gly LeuGln Arg Ile Phe Tyr Leu 565 570 575 Lys Leu Glu Asp Leu Val Pro Pro ProPro Ile Ile Asp Lys Ile Phe 580 585 590 Met Asp Thr Leu Pro Phe 595 3624 DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 36 gtgatattta cctccaaatg ccag 24

What is claimed is:
 1. A method of inhibiting proliferation of ahematopoietic cell, comprising the step of modulating the level of nor-1and/or nur77 nuclear receptor.
 2. The method of claim 1, wherein thehematopoietic cell is a hematopoietic stem cell.
 3. The method of claim1, wherein the hematopoietic cell is a hematopoietic myeloid cell. 4.The method of claim 1, wherein the modulating step is defined asincreasing the level of a nor-1 and/or nur77 nuclear receptorpolypeptide.
 5. The method of claim 4, wherein the increasing step isdefined as increasing the level of a nor-1 and/or nur77 nuclear receptorpolynucleotide.
 6. The method of claim 5, wherein the nor-1 and/or nur77nuclear receptor polynucleotide is increased through administration of avector comprising the polynucleotide.
 7. The method of claim 6, whereinthe vector is a viral vector or a non-viral vector.
 8. The method ofclaim 7, wherein the viral vector is an adenoviral vector, a retroviralvector, or an adeno-associated vector.
 9. The method of claim 7, whereinthe viral vector is an adenoviral vector.
 10. The method of claim 7,wherein the non-viral vector is a plasmid.
 11. The method of claim 5,wherein the nor-1 and/or nur77 nuclear receptor polynucleotide isincreased through upregulation of expression.
 12. The method of claim11, wherein the upregulation of expression is of the nor-1 and/or nur77nuclear receptor.
 13. The method of claim 12, wherein the upregulationof expression of the nor-1 and/or nur77 nuclear receptor is throughadministration of growth factors, cytokines, cyclic AMP, or a mixturethereof.
 14. The method of claim 1, wherein the cell is in a mammalafflicted with leukemia.
 15. A method of inhibiting proliferation of ahematopoietic cell, comprising the step of modulating the activity of anor-1 and/or nur77 nuclear receptor.
 16. The method of claim 15, whereinthe hematopoietic cell is a hematopoietic stem cell.
 17. The method ofclaim 15, wherein the hematopoietic cell is a hematopoietic myeloidcell.
 18. The method of claim 15, wherein the modulating step is definedas increasing transcriptional activity of a nor-1 and/or nur77 nuclearreceptor polypeptide.
 19. The method of claim 15, wherein the modulatingstep is further defined as administering an agonist to the nor-1 and/ornur77 nuclear receptor polypeptide.
 20. A method of treating leukemia inan individual, comprising the step of modulating a nor-1 and/or nur77nuclear receptor in said individual.
 21. The method of claim 20, whereinsaid modulating step occurs in a hematopoietic cell of the individual.22. The method of claim 21, wherein the hematopoietic cell is ahematopoietic stem cell.
 23. The method of claim 21, wherein thehematopoietic cell is a hematopoietic myeloid cell.
 24. The method ofclaim 20, wherein the modulating step is further defined as increasingthe activity of a nor-1 and/or nur77 nuclear receptor polypeptide. 25.The method of claim 20, wherein the modulating step is further definedas increasing the level of a nor-1 and/or nur77 nuclear receptorpolypeptide.
 26. The method of claim 20, wherein the modulating step isfurther defined as increasing the level of a nor-1 and/or nur77 nuclearreceptor polynucleotide.
 27. The method of claim 24, wherein theincreasing activity step is further defined as introducing an agonist tosaid nor-1 and/or nur77 nuclear receptor polypeptide.
 28. The method ofclaim 27, wherein the introducing step is further defined asadministering said agonist in a pharmaceutically acceptable compositionto said individual.
 29. The method of claim 27, wherein the agonist is aligand of said nor-1 and/or nur77 nuclear receptor.
 30. The method ofclaim 27, wherein the agonist is not a ligand of said nor-1 and/or nur77nuclear receptor.
 31. The method of claim 26, wherein the increasing thelevel of a nor-1 and/or nur77 nuclear receptor polynucleotide step isdefined as increasing expression of a respective nor-1 and/or nur77nuclear receptor in a cell of the individual.
 32. The method of claim31, wherein the cell is a hematopoietic bone marrow stem cell.
 33. Themethod of claim 31, wherein the cell is a hematopoietic myeloid cell.34. The method of claim 26, wherein the increasing the level of a nor-1and/or nur77 nuclear receptor polynucleotide step is defined asincreasing the half-life of a respective nor-1 and/or nur77 nuclearreceptor mRNA in a cell of the individual.
 35. The method of claim 34,wherein the cell is a hematopoietic bone marrow stem cell.
 36. Themethod of claim 34, wherein the cell is a hematopoietic myeloid cell.37. The method of claim 32, wherein the method further comprises thestep of administering said cell to an individual.
 38. The method ofclaim 33, wherein the method further comprises the step of administeringsaid cell to an individual.
 39. The method of claim 35, wherein themethod further comprises the step of administering said cell to anindividual.
 40. The method of claim 36, wherein the method furthercomprises the step of administering said cell to an individual.
 41. Amethod of increasing the level of a nor-1 and/or nur77 nuclear receptorin a hematopoietic cell, comprising the step of administering a compoundto the cell to increase the expression of said nor-1 and/or nur77nuclear receptor.
 42. The method of claim 41, wherein said compound is agrowth factor, cytokine, cyclic AMP, or a mixture thereof.
 43. Themethod of claim 41, wherein said method is further defined asadministering said compound in a pharmaceutically acceptable compositionto said individual.
 44. A method of identifying an upregulator ofexpression of a nor-1 and/or nur77 nuclear receptor, comprising thesteps of: introducing to a cell a test agent, wherein the cell comprisesa marker sequence and wherein the expression of the marker sequence isregulated by a nor-1 and/or nur77 nuclear receptor regulatory sequence;and measuring for an increase in the expression level of the markersequence, wherein when said increase occurs following introduction ofsaid test agent to said cell, said test agent is said upregulator. 45.The method of claim 44, wherein the method further comprisesadministering the upregulator in a pharmaceutically acceptablecomposition to an individual.
 46. The method of claim 45, wherein theindividual is susceptible to leukemia or is diagnosed with leukemia. 47.A method of identifying a compound for the treatment of leukemia,comprising the steps of: obtaining a compound suspected of havingactivity of a nor-1 and/or nur77 nuclear receptor agonist; anddetermining whether said compound has said activity.
 48. The method ofclaim 47, wherein the agonist is a ligand of a nor-1 and/or nur77nuclear receptor.
 49. The method of claim 47, wherein the method furthercomprises: dispersing the compound in a pharmaceutical carrier; andadministering a therapeutically effective amount of the compound in thecarrier to an individual having leukemia.
 50. As a composition ofmatter, the compound obtained by the method of claim
 47. 51. Apharmacologically acceptable composition comprising: the compoundobtained by the method of claim 47; and a pharmaceutical carrier.
 52. Amethod of screening for a compound for the treatment of leukemia,comprising the steps of: providing a first vector comprising a nor-1 ornur77 nucleic acid sequence encoding a respective nor-1 or nur77 geneproduct, wherein the expression of said nor-1 or nur77 nucleic acidsequence is under the control of a first regulatory sequence; providinga second vector comprising a reporter nucleic acid sequence encoding areporter gene product, wherein the expression of said reporter nucleicacid sequence is under the control of a second regulatory sequence,wherein the second regulatory sequence is responsive to nor-1 or nur77;providing a test agent; providing a leukemia cell line, wherein cells insaid cell line comprise conditions suitable for expression of said nor-1or nur77 gene product and said reporter gene product; and assayingtranscriptional regulation activity of said nor-1 or nur77 gene productby measuring expression or activity of the reporter gene product in thepresence of said test agent, wherein when the expression or activity ofthe reporter gene product changes in the presence of the test agent, thetest agent is the compound for the treatment of leukemia.
 53. The methodof claim 52, wherein the leukemic cell line is K562, U937, AML-193,HL-60, LSTRA, or CEM.
 54. The method of claim 52, wherein the firstvector, second vector, test agent, or a combination thereof areintroduced into the cell line.
 55. The method of claim 52, wherein thereporter nucleic acid is β-galactosidase, green fluorescent protein,blue fluorescent protein, or chloramphenicol acetyltransferase.
 56. Themethod of claim 52, wherein the expression or activity of the reportergene product increases in the presence of the test agent.
 57. A mousemodel for leukemia, comprising a mouse having defective nor-1 and nur77nucleic acid sequences.
 58. The mouse model of claim 57, wherein themouse is further defined as having a knockout mutation in the genesencoding nor-1 and nur77, respectively.
 59. The mouse model of claim 57,wherein the mouse is further defined as having the nor-1KO/nur77±genotype, the nor-1±/nur77 KO, or the nor-1KO/nur77KO, wherein KO isdefined as a knockout.
 60. The mouse model of claim 57, wherein themouse comprises at least one symptom of leukemia.